Compositions of hsp60 peptides and viral antigens for vaccination and diagnosis

ABSTRACT

The present invention provides improved vaccines comprising an isolated viral antigenic peptide and a synthetic peptide derived from a T cell epitope of HSP60. The invention includes mixtures where the peptide serves as an adjuvant as well as conjugates where the peptide is covalently linked to the viral antigen. The known synthetic peptide carrier, p458, provides significantly improved immunogenicity for synthetic viral epitopes and analogs. Ec27 is a novel peptide derived from HSP60 which increases the immunogenicity substantially of the viral antigen both as a mixture or a covalent conjugate. Some of the isolated viral epitopes are novel and are claimed for diagnostic as well as therapeutic or prophylactic uses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/164,411, filed Jun. 20, 2011, which is a divisional of U.S. patentapplication Ser. No. 11/908,474, filed May 15, 2008, which is U.S.national stage of PCT/IL2006/000222, filed Feb. 21, 2006, which is basedon and claims the benefit of U.S. Provisional Patent Application No.60/661,017, filed Mar. 14, 2005, the contents of each of which isexpressly incorporated herein in its entirety by this reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 32,202 byte ASCII (text) file named“Seq_List” created on Jan. 2, 2014.

FIELD OF THE INVENTION

The present invention relates to vaccines providing enhancedimmunogenicity comprising HSP60 peptides conjugated to or mixed with aviral antigen. The present invention further identifies certain novelepitopes, compositions thereof and methods of using same for vaccinationor diagnosis.

BACKGROUND OF THE INVENTION

Despite remarkable achievements in the development of vaccines forcertain viral infections (i.e., polio and measles), and the eradicationof specific viruses from the human population (e.g., smallpox), viraldiseases remain as important medical and public health problems. Indeed,viruses are responsible for several “emerging” (or re-emerging) diseases(e.g., West Nile encephalitis and Dengue fever), and viral infection isa cause of significant morbidity and mortality worldwide.

The presence of adequate T-cell help is important for the constructionof potent vaccines. Vaccines that induce both helper T cells and CTLsmay be more effective that those that induce CTLs only. Indeed, theimportance of cooperation between CD4⁺ and CD8⁺ T cells is emphasized inthe therapeutic vaccination against chronic viral infection (Zajac etal., 1998; Matloubian et al., 1994).

Classically, vaccines are manufactured by introducing killed orattenuated organisms into the host along with suitable adjuvants toinitiate the normal immune response to the organisms while, desirably,avoiding the pathogenic effects of the organism in the host. Theapproach suffers from the well known limitations in that it is rarelypossible to avoid the pathogenic response because of the complexity ofthe vaccine which includes not only the antigenic determinant ofinterest but many related and unrelated deleterious materials, anynumber of which may, in some or all individuals, induce an undesirablereaction in the host.

For example, vaccines produced in the classical way may includecompeting antigens which are detrimental to the desired immune response,antigens which include unrelated immune responses, nucleic acids fromthe organism or culture, endotoxins and constituents of unknowncomposition and source. These vaccines, generated from complexmaterials, inherently have a relatively high probability of inducingcompeting responses even from the antigen of interest.

HSP60 belongs to a family of chaperone molecules highly conservedthroughout evolution; a similar HSP60 molecule is present in all cells,prokaryotes and eukaryotes. The human HSP60 molecule was formerlydesignated HSP65, but is now designated HSP60 in view of more accuratemolecular weight information; by either designation, the protein is thesame. Apparently, no cell can exist without the ability to expressHSP60. Mammalian HSP60 is highly homologous to the bacterial cognates,showing about 50% amino acid identity (Jindal et al., 1989). Thus, HSP60is shared by the host and its parasites, and is immunogenic,cross-reactive, and universally expressed in inflammation. Furthermore,HSP60 is well recognized by the immune system (Konen Waisman et al.,1999, Konen Waisman et al., 1995) and is a part of the set ofself-molecules for which autoimmunity naturally exists; HSP60 is memberof the immunologic homunculus (Cohen, 1992). Heat shock, IFNγ, bacterialor viral infection, and inflammation, all result in the presentation ofendogenous HSP60 epitopes on MHC class II molecules leading to theactivation of HSP60-specific T cells, even in healthy individuals(Anderton et al., 1993; Hermann et al., 1991; Koga et al., 1989).

European Patent EP 262 710 and U.S. Pat. No. 5,154,923 describe peptideshaving an amino acid sequence corresponding to positions 171-240 and172-192, respectively, of a Mycobacterium boris BCG 64 kD polypeptide,that are useful as immunogens inducing resistance to autoimmunearthritis and similar autoimmune diseases.

PCT Patent Application No. WO 90/10449 describes a peptide designatedp277 having an amino acid sequence corresponding to positions 437-460 ofthe human HSP65 molecule that is useful as immunogen inducing resistanceto insulin dependent diabetes mellitus (IDDM). A control peptide,designated p278, corresponding to positions 458-474 of human HSP65, didnot induce resistance to IDDM.

Lussow et al. (1990) showed that the priming of mice with liveMycobacterium tuberculosis var. bovis (BCG) and immunization with therepetitive malaria synthetic peptide (NANP)₄₀ conjugated to purifiedprotein derivative (PPD), led to the induction of high and long-lastingtiters of anti-peptide IgG antibodies. Later on, Lussow et al. (1991)showed that mycobacterial heat-shock proteins (HSP) of 65 kDa(GroEL-type) and 70 kDa (DnaK-type) acted as carrier molecules in mice,previously primed with Mycobacterium tuberculosis var. boris (bacillusCalmette-Guerin, BCG), for the induction of high and long-lasting titersof IgG against the repetitive malaria synthetic peptide (NANP)₄₀.Anti-peptide antibodies were induced when the malaria peptide,conjugated to the mycobacterial HSP, was given in the absence of anyadjuvants.

Barrios et al. (1992) have shown that mice immunized with peptides oroligosaccharides conjugated to the 70 kDa HSP produced high titers ofIgG antibodies in the absence of any previous priming with BCG. Theanti-peptide antibody response persisted for at least 1 year. Thisadjuvant-free carrier effect of the 70 kDa HSP was T cell dependent,since no anti-peptide nor anti-70 kDa IgG antibodies were induced inathymic nu/nu mice. Previous immunization of mice with the 65 kDa or 70kDa HSP did not have any negative effect on the induction ofanti-peptide IgG antibodies after immunization with HSP-peptideconjugates in the absence of adjuvants. Furthermore, preimmunizationwith the 65 kDa HSP could substitute for BCG in providing effectivepriming for the induction of anti-(NANP)₄₀ antibodies. Finally, both the65 kDa and 70 kDa HSP acted as carrier molecules for the induction ofIgG antibodies to group C meningococcal oligosaccharides, in the absenceof adjuvants, suggesting that the use of HSPs as carriers in conjugatedconstructs for the induction of anti-peptide and anti-oligosaccharideantibodies could be of value in the design of new vaccines for eventualuse in humans.

U.S. Pat. No. 5,736,146 discloses conjugates of poorly immunogenicantigens with a synthetic peptide carrier comprising a T cell epitopederived from the sequence of human heat shock protein HSP65, or ananalog thereof, said peptide or analog being capable of increasingsubstantially the immunogenicity of the poorly immunogenic antigen. The'146 patent discloses conjugates of a peptide corresponding to positions458-474 and 437-453 of human or mouse HSP60 and homologs thereof with awide variety of antigens including peptides, proteins andpolysaccharides such as bacterial polysaccharide (e.g. capsularpolysaccharide (CPS) Vi of Salmonella typhi), and antigens derived fromHIV virus or from malaria antigen.

U.S. Pat. No. 5,869,058 discloses conjugates of poorly immunogenicantigens, e.g., peptides, proteins and polysaccharides, with a syntheticpeptide carrier comprising a T cell epitope derived from the sequence ofE. coli HSP65 (GroEL), or an analog thereof, said peptide or analogbeing capable of increasing substantially the immunogenicity of thepoorly immunogenic antigen. A suitable peptide according to theinvention is Pep278e, which corresponds to positions 437-453 of the E.coli HSP65 molecule.

Human cytomegalovirus (HCMV) is a ubiquitous double-stranded DNA virusfrom the betaherpesvirus group; it is endemic in all human populations.In North America, HCMV infects about 50% of the population outside ofurban centers and up to 90% of the population within cities. HCMVdisease presents two major medical problems: first, it is the mostcommon congenital viral infection, causing birth defects includingmal-development of the central nervous system; up to 25% of asymptomaticinfected infants will develop neurologic sequelae. Second, HCMV becomesre-activated in immunocompromised patients.

A self-limiting acute phase of viral infection, persistent and latentphases normally characterize the pathogenesis of HCMV infection in theimmunocompetent host. The clinical outcome of HCMV infection isdetermined by the ability of infected individuals to mount protectivehumoral and T-cell mediated immune responses. In immunocompromisedhosts, including persons with HIV infection, cancer patients andallograft recipients, primary HCMV infection or reactivation of a latentvirus results in multi-organ HCMV disease, associated with high rates ofmorbidity and mortality. These grave clinical consequences emphasize theneed for effective HCMV vaccines to prevent not only primary infectionbut also to limit or prevent reactivation.

At present there is no protective vaccine available for CMV. Currentlyavailable antiviral drugs which target viral DNA replication areefficacious but exhibit significant host toxicity and a high spontaneousresistance rate.

West Nile virus is a member of the alpha-like Flaviviridae. TheFlavivirus genome is a single-stranded, positive-sense RNA approximately11 kb in length, containing a 5′untranslated region (5′UTR); a codingregion encoding the three viral structural proteins; seven nonstructuralproteins, designated NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5; and a3′untranslated region (3′UTR). The viral structural proteins include thecapsid (C), premembrane/membrane (prM) and envelope (E) proteins. Thestructural and nonstructural proteins are translated as a singlepolyprotein. The polyprotein is then processed by cellular and viralproteases.

West Nile virus affects birds as well as reptiles and mammals, togetherwith man. The West Nile virus is transmitted to birds and mammals by thebites of certain mosquitoes (e.g. Culex, Aedes, Anopheles). Directtransmission may happen from WNV infected subject to healthy subject byoral transmission (prey and transmission through colostrum) andblood/organ vectored transmission. Widespread in Africa, the geographicrange of WNV now also includes Australia, Europe, the Middle East, WestAsia and the USA. West Nile virus can cause a harsh, self-limitingfever, body aches, brain swelling, coma, paralysis, and death.

There is no effective treatment for the disease. A number of differentWNV vaccines are now in various stages of development and testing(Monath, 2001; Pletnev et al., 2003; Tesh et al., 2002; Hall et al.,2003), but presently a licensed human vaccine is not available for itsprevention. The only currently effective way to provide immediateresistance to WNV is by passive administration of protective antibodies(Casadevall, 2002). Mosquito control is currently considered thepractical strategy to combat the spread of disease, but effectivespraying is difficult to perform in urban areas. Clearly, an effectivevaccine is needed to protect at-risk populations.

There remains a need for improved vaccines conferring protection againstviral infections, using isolated epitopes. Furthermore, isolatedepitopes are needed for improved diagnostic tests.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods suitable forvaccination against and diagnosis of viral infections. According to someaspects the present invention provides a vaccine comprising an isolatedviral antigenic peptide and a peptide comprising a T cell epitope ofHSP60, wherein the HSP60 peptide enhances the immunogenicity of theviral antigenic peptide by at least two fold compared to the peptidewithout the HSP60 peptide. In certain currently preferred embodimentsthe immunogenicity is enhanced by at least 4-5 fold. Novel viral peptideantigens useful in vaccination and diagnosis are also provided.

In certain embodiments the vaccine compositions comprise a T cellepitope of HSP60 suitable to enhance the immunogenicity when used as anadjuvant peptide that is mixed with the viral antigen. In alternativeembodiments the vaccine comprises a T cell epitope of HSP60 suitable toenhance the immunogenicity of the viral antigenic peptide when used inconjugates where the HSP60 peptide is covalently linked to the viralantigenic peptide.

The enhanced immunogenicity of said viral antigen is measured by atleast one of the following: serum titer of antibodies directed to saidviral antigen; T cell proliferation in the presence of said viralantigen; cytokine secretion induced by said viral antigen; specific Tcell mediated lysis of virus-infected cells; and reduction of detectableviral load.

According to another aspect, the invention provides conjugatescomprising a viral antigen covalently attached to a synthetic peptidecarrier comprising a T cell epitope of HSP60. According to someembodiments, the synthetic peptide carrier is the known peptide carrierp458, a Major Histocompatibility Complex (MHC) class II-restrictedpeptide derived from murine HSP60 (aa 458-474, also designatedpreviously as p278m), or an analog or derivative thereof. In otherembodiments, the synthetic peptide carrier is Ec27, a novel peptidederived from E. coli HSP60 (GroEL, aa 391-410).

According to the present invention, it is now disclosed that conjugatescomprising a synthetic peptide carrier selected from p458 and Ec27covalently attached to a viral antigen are unexpectedly effective inconferring immunity against viral infections. It is now demonstrated forthe first time that these conjugates significantly enhance effectiveimmunity against both DNA and RNA viruses, latent and acute infections,and when combined with CTL-, B cell- and MHC II-restricted viralepitopes.

The principles of the invention are exemplified by two model systems forviral infections. Mouse Cytomegalovirus (MCMV) infection in mice is anestablished model system for examining human infection with HumanCytomegalovirus (HCMV), a DNA virus which is characterized by a latentinfection following a self-limiting acute phase of viral infection. WestNile virus (WNV) infection in mice serves as a model for the acute viralinfection of WNV in humans.

According to a some embodiments, the present invention provides aconjugate comprising a viral antigen covalently attached to a syntheticpeptide carrier comprising a T cell epitope of HSP60 in which saidsynthetic peptide carrier is selected from the group of peptidesconsisting of:

-   -   (a) NEDQKIGIEIIKRTLKI (p458h; SEQ ID NO: 1),    -   (b) NEDQKIGIEIIKRALKI (p458; SEQ ID NO:2),    -   (c) EGDEATGANIVKVALEA (p458mt; SEQ ID NO:3),    -   (d) NEDQNVGIKVALRAMEA (p458e; SEQ ID NO:4),    -   (e) an analog of p458h (SEQ ID NO: 1) that has at least 70% of        the electric and hydrophilicity/hydrophobicity characteristic of        human HSP60 from position 458 to position 474, said peptide or        analog being capable of increasing substantially the        immunogenicity of the viral antigen when the conjugate is        administered in vivo, and derivatives thereof,

(f) KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76) and analogs, fragments andderivatives thereof.

In one embodiment, the synthetic peptide is an analog of p458h (SEQ IDNO: 1): ⁴⁵⁸NEDQKIGIEIIKRTLKI⁴⁷⁴ in which the residue E⁴⁵⁹ is either E orD; the residue D⁴⁶⁰ is either D or E; the residue K⁴⁶² is either K or Ror ornithine (Orn); the residue I⁴⁶³ is either I or L, V, M, F,norleucine (Nle) or norvaline (Nva); the residue I⁴⁶⁵ residue is eitherI or L, V, M, F, Nle or Nva; the residue E⁴⁶⁶ is either E or D; theresidue I⁴⁶⁷ is either I or L, V, M, F, Nle or Nva; the residue I⁴⁶⁸ iseither I or L, V, M, F, Nle or Nva; the residue K⁴⁶⁹ is either K or R orOrn; the residue R⁴⁷⁰ is either R, K or Orn; the residue L⁴⁷² in eitherL or I, V, M, F, Nle or Nva; the residue K⁴⁷³ is either K or R or Orn;and the residue I⁴⁷⁴ is either I or L, V, M, F, Nle or Nva.

In another aspect, there is provided a novel adjuvant peptide derivedfrom E. coli HSP60 (GroEL) protein, useful for the compositions andmethods of the invention. The novel adjuvant peptide, herein designatedEc27, has an amino acid sequence corresponding to positions 391-410 ofGroEL (corresponding to accession number gi:45686198 without the firstmethionine residue, SEQ ID NO:83), as follows: KKARVEDALHATRAAVEEGV (SEQID NO:76). It is to be explicitly understood that the correspondingpeptides from mammalian species are included within the scope of thepresent invention. The corresponding human peptide exhibits 80%homology, having the sequence set forth in SEQ ID NO:86, as follows:KKDRVTDALNATRAAVEEGI (Ec27h). Ec27 analogs, fragments, derivatives,conjugates and salts are also contemplated by the present invention.

The Ec27 peptide is now demonstrated for the first time to increasesignificantly the immunogenicity of a broad array of antigens, includingbut not limited to viral antigens, bacterial antigens and mammalianantigens, e.g., viral peptide antigens, bacterial polysaccharides andantibodies. Surprisingly Ec27 was found to increase the immunogenicityof antigens when covalently conjugated to the antigen, as well as whenmixed with the antigen. Unexpectedly, Ec27 could even further increasethe immunogenicity of antigens of the invention conjugated to the p458carriers.

In another embodiment, the invention further provides vaccinecompositions comprising an antigen and a peptide adjuvant having anamino acid sequence as set forth in SEQ ID NO:76 or an analog, fragmentor derivative thereof. In various embodiments, the antigen is selectedfrom the group consisting of: a peptide, a peptide derivative, aprotein, a polysaccharide (e.g. a bacterial polysaccharide), and anantibody. In one embodiment, the vaccine composition comprises aconjugate of the peptide adjuvant and said antigen. In alternateembodiments, said vaccine composition comprises an admixture of saidpeptide adjuvant and said antigen.

In another aspect, the viral antigen used in the conjugates andcompositions of the invention comprises at least one epitope selectedfrom: a CTL epitope (a MHC I restricted T cell epitope), a B cellepitope and a MHC II restricted T cell epitope.

The viral antigen used in the conjugates of the invention may be derivedfrom any virus of interest. In certain embodiments, the virus belongs tothe herpesviridae family. In other particular embodiments, the virusbelongs to the betaherpesvirus subfamily. In one particular embodiment,the viral antigen is derived from immediate early gene 1 (IE-1) proteinof a virus belonging to herpesviridae. In another particular embodiment,the viral antigen comprises a CTL epitope. In another particularembodiment, the virus is CMV. In one preferred embodiment, the viralantigen is derived from immediate early gene 1 (IE-1) protein of CMV. Inanother preferred embodiment, the viral antigen comprises a CTL epitope.

In other embodiments, the virus belongs to the Flaviviridae family. Inother particular embodiments, the virus belongs to the flavivirus genus.According to various particular embodiments, the virus is selected fromthe group consisting of: West Nile virus (WNV), Yellow fever virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, Kunjinvirus, Japanese encephalitis virus, Dengue virus type 1, Dengue virustype 2, Dengue virus type 3 and Dengue virus type 4. In one particularembodiment, the viral antigen is derived from West Nile Virus (WNV).

In one preferred embodiment, the viral antigen is derived from theenvelope (E) protein of a virus belonging to the flaviviridae family. Inanother preferred embodiment, the viral antigen is derived from the E3domain of said protein. In another preferred embodiment, said viralantigen comprises a B cell epitope and a MHC II restricted epitope. Inanother preferred embodiment, the viral antigen is derived from the WNVenvelope (E) protein. In another preferred embodiment, the viral antigenis derived from the E3 domain of said protein. In another preferredembodiment, said viral antigen comprises a B cell epitope and a MHC IIrestricted epitope.

Other embodiments of the present invention are directed to novelisolated viral peptide antigens that may be used in conjugation with thecarriers of the invention for anti viral vaccination, as well as fordiagnostic purposes, as specified herein.

In another aspect, there is provided a novel peptide antigen derivedfrom WNV E3 domain of E protein, hereby designated p15, having an aminoacid sequence corresponding to positions 355-369 of the E protein.Depending on the particular strain of WNV, this novel antigen has anamino acid sequence selected from the group consisting of:LVTVNPFVSVATANS (SEQ ID NO:11) and LVTVNPFVSVATANA (SEQ ID NO:12). Otherembodiments are directed to analogs, homologs, fragments and derivativesthereof. In other embodiments, the invention provides proteins, peptidesand conjugates comprising said antigen. In one particular embodiment,the peptide has an amino acid sequence as set forth in any one of SEQ IDNOS:34-35 (see Table 1).

In other embodiments, there is provided a p15 homologous peptide antigenderived from the E3 domain of the envelope protein of a flavivirusselected from the group consisting of: West Nile virus (WNV), Yellowfever virus, St. Louis encephalitis virus, Murray Valley encephalitisvirus, Kunjin virus, Japanese encephalitis virus, Dengue virus type 1,Dengue virus type 2, Dengue virus type 3 and Dengue virus type 4. Incertain particular embodiments, the p15 homologous antigen has an aminoacid sequence as set forth in any one of SEQ ID NOS:25-33 and 36-44 (seeTable 1), and analogs, homologs, fragments, and derivatives thereof.

In another aspect, there is provided a second novel WNV peptide antigenderived from the E protein, herein denoted p17, having the followingamino acid sequence: YIVVGRGEQQINHHWHK (SEQ ID NO:21). Other embodimentsare directed to analogs, homologs, fragments, and derivatives thereof.

In other embodiments, the invention provides nucleic acid moleculesencoding said novel peptide antigens, recombinant constructs comprisingthese nucleic acid molecules, and vectors and cells comprising them.

In another embodiment, there are provided conjugates comprising asynthetic peptide carrier of the invention and a viral antigen having anamino acid sequence as set forth in any one of SEQ ID NOS:11-12 and34-35 and analogs, homologs, fragments and derivatives thereofcovalently attached to a synthetic peptide carrier of the invention. Inanother embodiment, the conjugate has an amino acid sequence as setforth in any one of SEQ ID NOS:13-16, 65-66 and 77-78. In otherembodiments, the conjugates of the invention comprise a viral antigenhaving an amino acid sequence as set forth in any one of SEQ IDNOS:25-33 and 36-44 covalently attached to a synthetic peptide carrierof the invention. In another embodiment, the conjugate has an amino acidsequence as set forth in any one of SEQ ID NOS:56-64, and 67-75. Inother embodiments, the conjugates of the invention comprise a viralantigen having an amino acid sequence as set forth in SEQ ID NO:21covalently attached to a synthetic peptide carrier of the invention. Inanother embodiment, the conjugate has an amino acid sequence as setforth in any one of SEQ ID NOS:23-24 and 79 (see Table 4).

In another aspect, the invention provides vaccine compositionscomprising the conjugates of the invention and a pharmaceuticallyacceptable carrier, adjuvant, excipient or diluent. In another aspect,the invention provides vaccine compositions comprising a viral antigenin admixture with Ec27 and a pharmaceutically acceptable carrier,adjuvant, excipient or diluent. In another aspect, the inventionprovides vaccine compositions comprising the novel isolated viralpeptide antigens of the invention and a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

In yet another aspect, the invention provides methods for increasing theimmunogenicity of a viral antigen which comprises linking the antigen toa synthetic peptide carrier of the invention.

In another aspect, the invention provides methods for immunizing asubject in need thereof against a viral infection, comprisingadministering to the subject an effective amount of a vaccinecomposition comprising a conjugate of the invention and apharmaceutically acceptable carrier, adjuvant, excipient or diluent.

The vaccine composition may be administered to said subject before theexposure of said subject to the virus or after exposure of said subjectto said virus.

In another aspect, the invention provides methods comprising:

-   -   (a) isolating a viral antigen, comprising at least one epitope        selected from: a CTL epitope, a B cell epitope and a MHC        II-restricted epitope;    -   (b) conjugating said viral antigen to a synthetic peptide        carrier of the invention to form a peptide-carrier conjugate;        and    -   (c) administering to the subject an effective amount of a        vaccine composition comprising the conjugate and a        pharmaceutically acceptable carrier, adjuvant, excipient or        diluent.

According to various embodiments, the compositions and methods of theinvention are suitable for vaccinating a subject selected from a groupconsisting of: humans, non-human mammals and non-mammalian animals. In apreferred embodiment, the subject is human.

Other aspects of the present invention are directed to diagnostic kitsand methods utilizing the novel isolated viral peptide antigens fordetermining the exposure of a subject to a flavivirus.

In one aspect, there is provided a diagnostic kit comprising at leastone viral peptide antigen of the invention and means for detectingwhether the peptide antigen is bound specifically to a suitablebiological sample.

In another aspect, the invention provides methods for diagnosingexposure of a subject to a flavivirus and for diagnosing a flavivirusinfection in a subject, comprising the steps of:

-   -   (a) contacting a suitable biological sample with a viral antigen        having an amino acid sequence as set forth in any one of SEQ ID        NOS:11-12, 25-44 and 21 and analogs, homologs, derivatives and        salts thereof under conditions such that an immune reaction can        occur;    -   (b) determining the extent of specific antigen binding to the        biological sample, wherein a level significantly higher than the        level obtained for a sample obtained from a non-infected subject        is indicative of exposure of the subject to the flavivirus.

In certain embodiments, the kits and diagnostic methods of the inventionare useful for the differential diagnosis of a flavivirus infection.

These and other embodiments of the present invention will becomeapparent in conjunction with the figures, description and claims thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Kinetics of MCMV infection in spleen and salivary gland (SG) ofBALB/c mice. Mice were challenged i.p. with 5×10⁴ pfu of MCMV. A.Infectious virus titers in spleen and salivary gland were measured atdifferent time points after infection (calculated as log 10 pfu/0.1 gtissue). The data represent the average of 5 experiments. B. PCRamplification of the 356 bp product of MCMV gB DNA in spleen andsalivary gland at different time points after infection. Results arefrom 1 representative experiment of 3 performed.

FIG. 2. The effectiveness of the p458-89pep vaccine. A. The experimentaldesign. B. Infectious MCMV titers in salivary gland 14, 21 and 28 daysafter challenge. Data represent the average titer (±SE) of the salivaryglands of 3 individual mice of each group. In salivary glands of miceimmunized with p458-89pep, virus titers on day 28 (asterisks) were belowdetection (i.e. <2 log₁₀ pfu/0.1 gr tissue). C. PCR amplification of the356 bp product of MCMV gB. Template DNA was extracted from salivaryglands of immunized mice on day 28 after MCMV challenge. Immunizationwith: a. IFA only without challenge; b. IFA only; c. 89pep; d.p458-89pep; e. control-89pep; f. PCR mix without template DNA (negativePCR control). Results are from 1 representative experiment of 2performed.

FIG. 3. IFNγ secretion from spleen and salivary gland (SG) cell culturesof MCMV infected mice. Spleen cell (FIG. 3A) and fractionated salivarygland mononuclear cell cultures (FIG. 3B) were prepared on differentdays after virus infection as described in methods. IFNγ secretion insupernatant was measured by ELISA after 3 days culture with (squares) orwithout (circles) 89pep stimulation (10 μg/ml) in vitro. Data representthe average (±SE) of 3 experiments.

FIG. 4. IFNγ levels from spleen cell cultures after vaccination withp458-89pep. Spleen cell cultures were prepared 10 days after vaccinationwith the various peptides or after challenge of naïve mice with MCMV. Acontrol group received IFA without any peptide. IFNγ secretion insupernatants was measured by ELISA after 3 days stimulation in vitrowith p458, 89pep (10 μg/ml), or without stimulation (No-stim). Datarepresent the average of 6 experiments (±SE). * p≦0.05 compared top458-89pep, two-tailed T-test.

FIG. 5. IFNγ-positive spleen cells after vaccination with p458-89pep.Spleen-cell cultures were prepared 7 days after vaccination with variouspeptides, or after challenge of naïve mice with MCMV. After 5 days ofstimulation in vitro with 89pep or without stimulation (No-stim), thecells were stained for CD4 (FIG. 5A) or CD8 (FIG. 5B) markers and forIFNγ. Numbers (27.43, 9.89 and 0.41) are percentage of IFNγ⁺ cells intotal CD8⁺ cells. Results are from 1 representative experiment of 2performed.

FIG. 6. CTL activity in spleen cell cultures after vaccination withp458-89pep. Spleen-cell cultures were prepared 7 days after immunizationwith various peptides or after challenge of naïve mice with MCMV. Acontrol group received IFA without any peptide. CTL activity wasmeasured after 6 days of stimulation in vitro with 89pep (10 μg/ml).Target cells were P815 pulsed with the 89pep (1 μg/ml). E:T ratio is25:1. The data represent the average (±SE) of 3 different experiments.

FIG. 7. IFNγ-positive salivary gland mononuclear cells 28 days afterMCMV challenge of vaccinated mice. Mice were vaccinated and thenchallenged with MCMV. Cells were stained for CD8 and for IFNγ.No-stimulation (No-stim) or stimulation with 89pep (89pep) relates tothe presence of 89pep during the 8 h incubation with golgi-stop step inthe ICCS protocol for IFNγ. Results are from 1 representative experimentof 2 performed.

FIG. 8. Recognition of peptides by IVIG-IL. Wells were coated with thedifferent peptides (1 μg/well) or with the WNV-Ag (1:700 dilution).After blocking and washing, IVIG-IL were added at 1:40 dilution andbinding was detected as described in methods (ELISA). Background ofELISA (no peptide at well, 0.078 OD to 0.120 OD in differentexperiments) was subtracted from each experimental point. Results arethe average of 4 independent ELISA experiments. Bars, ±SD.

FIG. 9. Recognition of peptides by serum from WNV-infected mice. Wellswere coated with the different peptides (1 μg/well) or with the WNV-Ag(1:700 dilution). After blocking and washing, naïve (gray columns) orWNV-infected (white columns) murine sera were added at 1:40 dilution andbinding was detected as described in methods (ELISA). Results are from 1representative experiment of 3 performed. Each experimental point wasperformed in triplicate. Bars, ±SD.

FIG. 10. Proliferation of splenocytes from WNV-infected mice followingin vitro stimulation with the different peptides. Mice were infectedwith 66 pfu of WNV. Six days after, spleens were harvested andsplenocytes were cultured with the different peptides (10 μg/ml) or withConA (5 μg/ml) for 3 days. Proliferation of cells derived from naïve(gray columns) or WNV-infected (white columns) mice was measured usingWST-1 method. Results are the average of 3 independent proliferationexperiments. Bars, ±SD.

FIG. 11. Anti-WNV Abs in the sera of mice immunized with differentpeptides or with WNV Ag. (A) Mice were immunized 3 times with thedifferent peptides or WNV-Ag at 7 day intervals. Seven days after the3^(rd) immunization mice were bled and sera were tested as follows.Wells were coated with WNV-Ag (1:700 dilution). After blocking andwashing, the different sera were added at 1:40 dilution and binding wasdetected as described in methods (ELISA). Results are the average of 4independent ELISA experiments. Bars, ±SD. (B) Isotypes of Anti-WNVantibodies.

FIG. 12. Proliferation of splenocytes from p32-immunized mice followingin vitro stimulation with the different peptides. Mice were immunized 3times with p32 at 7 day intervals. Seven days after the 3^(rd)immunization, spleens were harvested and splenocytes were cultured withthe different peptides or WNV-Ag (10 μg/ml) or with ConA (5 μg/ml) for 3days. Cell proliferation was measured using WST-1 method. Results arethe average of 3 independent proliferation experiments. Bars, ±SD.

FIG. 13. IFNγ secretion in the spleens of p32-vaccinated mice on day 7after immunization. Mice were immunized 3 times with p32 at 7 dayintervals. Seven days after the 3^(rd) immunization, spleens wereharvested and splenocytes were cultured with the different peptides orWNV-Ag (10 μg/ml) or with ConA (5 μg/ml) for 3 days. IFNγ levels in thesupernatants were measured as described in methods. Results are theaverage of 4 independent proliferation experiments. Bars, ±SD.

FIG. 14. p458-89pep reduces viral load of MCMV-infected mice. A. Theexperimental design. B. PCR amplification of the 363 bp product of MCMVIE-1.

FIG. 15. Viral loads following p32 immunization and WNV challenge.

FIG. 16. proliferation and IFN-γ secretion of splenocytes from miceimmunized by Ec27-p15 conjugates following in vitro stimulation withp15.

FIG. 17. Specific recognition of p15 (A, B) and p17 (B) by sera fromWNV-infected human patients.

FIG. 18. Proliferative response of BALB/c lymph node cells tooverlapping GroEL peptides after immunization with E. coli bacteria (A)or GroEL (B)

FIG. 19. Proliferative response of BALB/c lymph node cells toimmunization with the Ec27 peptide

FIG. 20. Proliferative response of different mouse strains toimmunization with the Ec27 peptide. BALB/c (A), BALB/k (B), and BALB/b(C) and SJL (D) mice were immunized s.c. with 20 mg of the Ec27 peptideemulsified in IFA. Ten days later lymph node cells (2×10⁵ cells perwell) were assessed for specific proliferation to the Ec27 peptide (fullcircles), the ec35 peptide (empty circles), or the acetylcholinereceptor peptide 259-271 (empty triangles). After 96 hours ofincubation, the ³H-thymidine incorporation was assessed as a measure ofproliferation. Results are shown as mean cpm of quadruplicate wells. Thestandard deviations are indicated.

FIG. 21. Adjuvant effect of the Ec27 peptide. (A) anti-PAb-246reactivity; (B) anti-p53 reactivity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel conjugates comprising a viralantigen covalently linked to a synthetic peptide carrier comprising a Tcell epitope of HSP60. The synthetic peptide carrier, p458, is a MHCclass II-restricted peptide derived from murine HSP60 (aa 458-474, alsodesignated previously as p278m), or an analog or derivative thereof,which peptide or analog being capable of increasing substantially theimmunogenicity of the viral antigen. In other embodiments, the carrieris Ec27, a novel peptide derived from E. coli GroEL (aa 391-410). Theinvention provides vaccine compositions comprising the conjugates of theinvention, and methods for their use in vaccinating a subject in needthereof against a viral infection. The invention further provides novelviral peptide antigens, conjugates and vaccine compositions thereof anduses thereof in vaccination and diagnosis.

The present invention discloses unexpectedly that a vaccine compositioncomprising a conjugate of a viral antigen and a peptide carrier derivedfrom HSP60 p458 or Ec27 is highly efficacious in conferring protectiveimmunity against a viral infection in vivo. It is now demonstrated forthe first time that p458 and Ec27 enhance effective immunity even forconjugates comprising antigens that are not poorly immunogenic. Thepeptide carriers of the invention were found to enhance theimmunogenicity of the viral antigen by at least two fold compared to thepeptide without the HSP60 peptide.

The present invention is based, in part, on studies of p458-viralantigen conjugate vaccination for the treatment of a chronic (latent)Cytomegalovirus (CMV) infection associated with persistence of virus inthe salivary glands. A conjugate comprising 89pep, an antigen derivedfrom murine CMV (MCMV) immediate early gene 1 protein (IE-1), fused top458, was more effective than the 89pep in inducing 89pep-specific IFNγsecretion and specific CTL activity. The p458-89pep chimeric peptideinduced sustained IFNγ secretion in the salivary gland specific to 89pepand only this immunization was associated with clearance of virus fromthe salivary gland.

The present invention is also based, in part, on studies of p458-viralantigen and Ec27-viral antigen conjugate vaccination against an acuteviral infection of West Nile Virus (WNV). A conjugate comprising p15, anovel antigen derived from WNV envelope (E) protein, fused to p458 wascapable, upon immunization, to significantly reduce the mortalityassociated with the infection, while immunization with p15 alone couldonly moderately affect the mortality rate. The conjugate was moreeffective than the viral antigen alone in inducing WNV-specificneutralizing antibodies as well as WNV-specific T cell proliferation andIFNγ secretion. Ec27-p15 conjugate was also more effective than p15alone in inducing p15-specific T cell proliferation and IFNγ secretion.

Thus, the conjugates of the invention are herein demonstrated to beeffective against both DNA and RNA viruses, latent and acute infections,and when combined with CTL-, B cell- and MHC II-restricted viralepitopes.

Ec27, a novel adjuvant peptide derived from E. coli HSP60 (GroEL)protein, was found to increase significantly the immunogenicity of abroad array of antigens, including but not limited to viral antigens,bacterial antigens and mammalian antigens, e.g., viral peptide antigens,bacterial polysaccharides and antibodies. Surprisingly Ec27 was found toincrease the immunogenicity of antigens when covalently conjugated tothe antigen, as well as when mixed with the antigen. Unexpectedly, Ec27could even further increase the immunogenicity of antigens conjugated tothe p458 carriers. Ec27 has an amino acid sequence corresponding topositions 391-410 of GroEL (corresponding to accession numbergi:45686198 without the first methionine residue, SEQ ID NO:83), asfollows: KKARVEDALHATRAAVEEGV (SEQ ID NO:76).

According to a first aspect, the present invention provides a conjugatecomprising a viral antigen covalently attached to a synthetic peptidecarrier comprising a T cell epitope of HSP60 in which said syntheticpeptide carrier is selected from the group of peptides consisting of:

-   -   (a) NEDQKIGIEIIKRTLKI (p458h, derived from human HSP60; SEQ ID        NO: 1),    -   (b) NEDQKIGIEIIKRALKI (p458, derived from mouse HSP60; SEQ ID        NO:2),    -   (c) EGDEATGANIVKVALEA (p458mt, derived from M. tuberculosis        HSP60; SEQ ID NO:3),    -   (d) NEDQNVGIKVALRAMEA (p458e, derived from E. coli HSP60; SEQ ID        NO:4. It should be noted, that the amino acid sequence of p458e        corresponds to positions 432-448 of SEQ ID NO:83)    -   (e) an analog of p458h (SEQ ID NO: 1) that has at least 70% of        the electric and hydrophilicity/hydrophobicity characteristic of        human HSP60 from position 458 to position 474, said peptide or        analog being capable of increasing substantially the        immunogenicity of the viral antigen when the conjugate is        administered in vivo,    -   (f) KKARVEDALHATRAAVEEGV (Ec27, derived from E. coli HSP60; SEQ        ID NO:76).

The active peptide carriers according to the invention are characterizedas being highly charged, i.e. of strong electric properties (7 out of 17constituent amino acid residues of p458 are either negatively orpositively charged) and highly hydrophobic (6 amino acid residues). Thepeptide p458h is further characterized as possessing a polarnegatively-charged N-terminal domain, a polar positively-chargedC-terminal domain and a highly hydrophobic core. These overall featuresshould be maintained in order to preserve efficacy. Thus, following theabove general outline certain amino acids substitution will lead toactive peptides. More specifically, positions 6, 8, 10, 11, 15 and 17 inthe p458 peptide chain (corresponding to positions 463, 465, 467, 468,472 and 474 of the human HSP60 molecule) can be occupied by either I orL or by other hydrophobic amino acids, natural, such as V, M, or F, orunnatural amino acids, such as norleucine (Nle) or norvaline (Nva).Positions 5, 12, 13 and 16 in the p458h chain (corresponding topositions 462, 469, 470 and 473 of the human HSP60 molecule) can beoccupied by either K or R or by unnatural positively charged aminoacids, such as ornithine (Orn). Interchange of E and D may also lead toactive derivatives.

With respect to the peptide carriers of the invention, the term“analogs” relates to peptides obtained by replacement, deletion oraddition of amino acid residues to the sequence, optionally includingthe use of a chemically derivatized residue in place of anon-derivatized residue, as long as they have the capability ofenhancing substantially the immunogenicity of viral antigen molecules.Analogs, in the case of p458, are peptides such that at least 70%,preferably 90-100%, of the electric properties and of the hydrophobicityof the peptide molecule are conserved. These peptides can be obtained,without limitation, according to the instructions in the paragraphhereinbefore. Ec27 analogs are preferably of at least about 70%, morepreferably of at least about 80-90% similarity in their amino acidsequence of Ec27. For example, the corresponding human peptide, havingthe sequence set forth in SEQ ID NO:86 (KKDRVTDALNATRAAVEEGI, Ec27h),exhibits 80% amino acid identity to Ec27.

The terms “covalently attached” and “conjugated” as used herein refer toa conjugate comprising an antigen and a synthetic peptide carrier linkedeither as a continuous fusion peptide or by means of chemicalconjugation (either directly or through a spacer), using methods wellknown in the art.

By “substantially increasing” the immunogenicity of a viral antigenmolecule it is meant to comprise both the induction of an increase inthe level of antibodies (Abs) against said antigen as well as thepresentation of said antibodies as mainly of the IgG isotype.Alternatively, the term may represent an increase in antigen-specific Tcell response, as measured either as increased CTL activity(antigen-dependent lysis) or as increased antigen-specific T cellproliferation or cytokine secretion (e.g. Thl cytokines such as IFNγ).Non-limitative examples for measuring the level of specific Abs andantigen-specific T cell response according to the invention arepresented in the Examples hereinbelow.

In another aspect, the viral antigen comprises at least one epitopeselected from: a CTL epitope (a MHC I restricted T cell epitope), a Bcell epitope and a MHC II restricted T cell epitope. Methods foridentifying suitable candidate epitopes are within the abilities ofthose of skill in the art (for example, without limitation, by usingepitope prediction software).

The viral antigen used in the conjugates of the invention may be derivedfrom any virus of interest. In certain embodiments, the virus belongs tothe herpesviridae family. This family includes, but is not limited to,human viruses such as human herpesvirus 1 (HHV-1, also known as herpessimplex virus 1, HSV1), HHV-2 (HSV2), HHV-3 (Varicella-zoster virus,VSV), HHV-4 (Epstein-Barr virus, EBV), HHV-5 (cytomegalovirus, CMV),HHV-6, HHV-7 and HHV-8.

In other particular embodiments, the virus belongs to thebetaherpesvirus subfamily (e.g. CMV and EBV). In another particularembodiment, the virus is CMV. In one preferred embodiment, the viralantigen is derived from immediate early gene 1 (IE-1) protein of aherpesvirus. In another preferred embodiment, the viral antigen isderived from immediate early gene 1 (IE-1) protein of a CMV. In anotherpreferred embodiment, the viral antigen derived from IE-1 proteincomprises a CTL epitope.

In other embodiments, the virus belongs to the Flaviviridae family. Thisfamily currently contains three genera, the flaviviruses (e.g.Tick-borne encephalitis viruses, Japanese encephalitis viruses, Dengue,Yellow fever and viruses such as Modoc and Uganda virus), thepestiviruses (e.g. bovine viral diarrhea, Border disease), and thehepatitis C viruses (e.g. hepatitis C virus, HCV).

In various embodiments, the virus is selected from the group consistingof: West Nile virus (WNV), Yellow fever virus, St. Louis encephalitisvirus, Murray Valley encephalitis virus, Kunjin virus, Japaneseencephalitis virus, Dengue virus type 1, Dengue virus type 2, Denguevirus type 3 and Dengue virus type 4. In one particular embodiment, theviral antigen is derived from West Nile Virus (WNV). In one preferredembodiment, the viral antigen is derived from the WNV envelope (E)protein. In another preferred embodiment, the viral antigen is derivedfrom the E3 domain of said protein. In another preferred embodiment,said viral antigen comprises a B cell epitope and a MHC II restrictedepitope.

In other embodiments, there is provided a novel antigen derived from WNVE3 domain of E protein, hereby designated p15, corresponding to aa355-369 of the E protein. In various embodiments, the antigen has anamino acid sequence as set forth in any one of SEQ ID NOS: 11 and 12(LVTVNPFVSVATANS and LVTVNPFVSVATANA, respectively). In otherembodiments, the invention provides proteins, peptides and conjugatescomprising said antigen. For example, without limitation, said antigenmay be conjugated with a peptide or lipid carrier or adjuvant.

In another embodiment, the conjugates of the invention comprise a viralantigen having an amino acid sequence as set forth in any one of SEQ IDNOS:11 and 12 covalently attached to a synthetic peptide carrier of theinvention. In another embodiment, the conjugate has an amino acidsequence as set forth in any one of SEQ ID NOS:13(NEDQKIGIEIIKRALKILVTVNPFVSVATANS), 14(NEDQKIGIEIIKRALKILVTVNPFVSVATANA), 15(NEDQKIGIEIIKRTLKILVTVNPFVSVATANS), 16(NEDQKIGIEIIKRTLKILVTVNPFVSVATANA), 77(KKARVEDALHATRAAVEEGVLVTVNPFVSVATANS), and 78(KKARVEDALHATRAAVEEGVLVTVNPFVSVATANA).

Other embodiments are directed to homologs, analogs, fragments andderivatives of p15, as detailed hereinbelow.

According to certain embodiments, the invention provides p15 homologsderived from a flavivirus, and active fragments and extensions thereof,as detailed in Table 1:

TABLE 1p15 homologous epitopes from various flaviviruses, active fragments andextensions thereof, and nucleotide sequences encoding them. nucleic acidsequence Virus Amino acid sequence (SEQ ID NO.) (SEQ ID NO.)West Nile virus LVTVNPFVSVATANS (11) 19 LVTVNPFVSVATANA (12) 20GRLVTVNPFVSVATANS (34) 54 GRLVTVNPFVSVATANA (35) 55 Yellow fever virusLVTVNPIASTNDDEVLIE (25) 45 GILVTVNPIASTNDDEVLIE (36)St. Louis encephalitis virus LVTVNPFISTGGANNKVM (26) 46GRLVTVNPFISTGGANNKVM (37) Murray Valley encephalitisMVTANPYVASSTANAKVL (27) 47 virus GRMVTANPYVASSTANAKVL (38) Kunjin virusLVTVNPFVSVSTANAKVL (28) 48 GRLVTVNPFVSVSTANAKVL (39)Japanese encephalitis virus LVTVNPFVATSSANSKVL (29) 49GRLVTVNPFVATSSANSKVL (40) Dengue virus type 1 LITANPIVTDKEKPVNIE (30) 50GRLITANPIVTDKEKPVNIE (41) Dengue virus type 2 LITVNPIVTEKDSPVNIE (31) 51GRLITVNPIVTEKDSPVNIE (42) Dengue virus type 3 LITANPVVTKKEEPVNIE (32) 52GRLITANPVVTKKEEPVNIE (43) Dengue virus type 4 IISSTPLAENTNSVTNIE (33) 53GRIISSTPLAENTNSVTNIE (44)

However, it should be understood that the amino acid sequence of thesehomologous epitopes may be altered in different variants and strains ofthese viruses. The present invention is thus further directed tohomologous peptides from different variants and strains of theseviruses.

With respect to the novel viral peptide antigens of the invention, theterm “analogs” relates to peptides obtained by replacement, deletion oraddition of amino acid residues to the sequence, optionally includingthe use of a chemically derivatized residue in place of anon-derivatized residue, as long as their ability to confer immunityagainst a viral infection when conjugated to the carriers of theinvention is retained. The term also includes homologs corresponding toamino acid sequences which are significantly related because of anevolutionary relationship, either between species (ortholog) or within aspecies (paralog). Peptide sequences having conserved amino acidsequence domains are examples of homologs. With respect to the novelviral peptide antigens of the invention, peptide homologs may have atleast about 40% identity in their amino acid sequence, preferably atleast 50%, more preferably at least about 70% and most preferably atleast about 90% identity. These values reflect the short length of thepeptides.

In another aspect, there is provided a second novel WNV epitope derivedfrom the E protein, herein denoted p17, having the following amino acidsequence: YIVVGRGEQQINHHWHK (SEQ ID NO:21). Other embodiments aredirected to analogs, homologs, fragments, and derivatives thereof.

In various embodiments, these peptides and homologs may be used inconjugation with the carriers of the invention. In certain particularembodiments, the conjugate has an amino acid sequence as set forth inany one of SEQ ID NOS: 23-24 and 56-75.

Peptide and Derivative Synthesis

The polypeptides and peptides of the invention may be synthesized usingany recombinant or synthetic method known in the art, including, but notlimited to, solid phase (e.g. Boc or f-Moc chemistry) and solution phasesynthesis methods. For solid phase peptide synthesis, a summary of themany techniques may be found in: Stewart and Young, 1963; andMeienhofer, 1973. For a review of classical solution synthesis, seeSchroder and Lupke, 1965.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the peptidesubstantially retains the desired functional property. Use of “D” aminoacids may be used as is known in the art to increase the stability orhalf-life of the resultant peptide.

Whenever p458 and Ec27 conjugates are mentioned in the invention, alsosalts and functional derivatives thereof are contemplated, as long asthey are able to substantially enhance the immunogenicity of the antigenmolecules. Thus, the present invention encompasses polypeptides orpeptides containing non-natural amino acid derivatives or non-proteinside chains.

The term derivative includes any chemical derivative of the polypeptidesor peptides of the invention having one or more residues chemicallyderivatized by reaction of side chains or functional groups. Suchderivatized molecules include, for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides, which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acidresidues. For example: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted or serine; andornithine may be substituted for lysine.

In addition, a peptide or conjugate can differ from the natural sequenceof the polypeptides or peptides of the invention by chemicalmodifications including, but are not limited to, terminal-NH₂ acylation,acetylation, or thioglycolic acid amidation, and byterminal-carboxlyamidation, e.g., with ammonia, methylamine, and thelike. Peptides can be either linear, cyclic or branched and the like,which conformations can be achieved using methods well known in the art.

It is noted that both shorter active fragments derived from the viralantigens denoted as SEQ ID NOS:11-12, 21 and 25-33 and longer peptidescomprising these sequences are within the scope of the presentinvention. Such fragments or peptides may be comprise, for example,peptides having 1-3 amino acids deleted at either termini, or additionof 1-3 amino acid residues or more from the flanking sequences of theviral protein to either termini, as long as their ability to conferimmunity against a viral infection when conjugated to the carriers ofthe invention is retained. It is to be understood that longer peptides,e.g. up to 50 amino acids in length may also be used for vaccinationaccording to the invention. However, shorter peptides are preferable, inone embodiment, for being easier to manufacture. Such extensions of thenovel peptide antigens of the present invention are not intended toinclude any known protein of fragment, such as the full length E3 domainof a flavivirus. The viral antigens, according to the present inventionare preferably 5-50 amino acids in length, more preferably 8-20 aminoacids in length. Exemplary fragments and extensions of the p15 andhomologs thereof according to the invention are presented in Table 1.

Addition of amino acid residues may be performed at either terminus ofthe polypeptides or peptides of the invention for the purpose ofproviding a “linker” by which the peptides of this invention can beconveniently bound to a carrier. Such linkers are usually of at leastone amino acid residue and can be of 40 or more residues, more often of1 to 10 residues. Typical amino acid residues used for linking aretyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.

The conjugates of the invention may also be created by means ofchemically conjugating a viral antigen with a p458 or Ec27 syntheticcarrier peptide, using methods well known in the art.

Nucleic Acids

In another aspect, the invention provides nucleic acid moleculesencoding the peptide antigens of the invention.

The nucleic acid molecules may include DNA, RNA, or derivatives ofeither DNA or RNA. An isolated nucleic acid sequence encoding a viralantigen or a HSP60 peptide can be obtained from its natural source,either as an entire (i.e., complete) gene or a portion thereof. Anucleic acid molecule can also be produced using recombinant DNAtechnology (e.g., polymerase chain reaction (PCR) amplification,cloning) or chemical synthesis. Nucleic acid sequences include naturalnucleic acid sequences and homologs thereof, including, but not limitedto, natural allelic variants and modified nucleic acid sequences inwhich nucleotides have been inserted, deleted, substituted, and/orinverted in such a manner that such modifications do not substantiallyinterfere with the nucleic acid molecule's ability to encode afunctional peptide of the present invention.

A nucleic acid molecule homolog can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., 1989). For example, nucleic acid molecules can be modified using avariety of techniques including, but not limited to, classic mutagenesistechniques and recombinant DNA techniques, such as site-directedmutagenesis, chemical treatment of a nucleic acid molecule to inducemutations, restriction enzyme cleavage of a nucleic acid fragment,ligation of nucleic acid fragments, polymerase chain reaction (PCR)amplification and/or mutagenesis of selected regions of a nucleic acidsequence, synthesis of oligonucleotide mixtures and ligation of mixturegroups to “build” a mixture of nucleic acid molecules and combinationsthereof. Nucleic acid molecule homologs can be selected from a mixtureof modified nucleic acids by screening for the function of the proteinencoded by the nucleic acid with respect to the induction of ananti-viral response, for example by the methods described herein.

A polynucleotide or oligonucleotide sequence can be deduced from thegenetic code of a protein, however, the degeneracy of the code must betaken into account. For example, an oligonucleotide having a nucleicacid sequence: ctggtgaccgtgaatccatttgtgtctgtggccacagccaactcg (SEQ IDNO:19) encodes a p15 antigen derived from West Nile Virus E protein:LVTVNPFVSVATANS (SEQ ID NO:11). However, nucleic acid sequences of theinvention also include sequences, which are degenerate as a result ofthe genetic code, which sequences may be readily determined by those ofordinary skill in the art. In other particular embodiments, the viralantigens of the invention are encoded by oligonucleotides having anucleic acid sequence as set forth in any one of SEQ ID NOS:20, 22 and45-55 (see Table 1).

The oligonucleotides or polynucleotides of the invention may contain amodified internucleoside phosphate backbone to improve thebioavailability and hybridization properties of the oligonucleotide orpolynucleotide. Linkages are selected from the group consisting ofphosphodiester, phosphotriester, methylphosphonate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoroanilidate,phosphoramidate, phosphorothioate, phosphorodithioate or combinationsthereof.

Additional nuclease linkages include alkylphosphotriester such asmethyl- and ethylphosphotriester, carbonate such as carboxymethyl ester,carbamate, morpholino carbamate, 3′-thioformacetal, silyl such asdialkyl(C1-C6)- or diphenylsilyl, sulfamate ester, and the like. Suchlinkages and methods for introducing them into oligonucleotides aredescribed in many references, e.g. reviewed generally by Peyman andUlmann, (1990).

The present invention includes a nucleic acid sequence of the presentinvention operably linked to one or more transcription control sequencesto form a recombinant molecule. The phrase “operably linked” refers tolinking a nucleic acid sequence to a transcription control sequence in amanner such that the molecule is able to be expressed when transfected(i.e., transformed, transduced or transfected) into a host cell.Transcription control sequences are sequences which control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those which controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences. Suitable transcription control sequences includeany transcription control sequence that can function in at least one ofthe recombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin animal, bacteria, helminth, insect cells, and animal cells.

A nucleic acid molecule of the invention may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Vectors can be introduced into cells or tissues by any one of a varietyof known methods within the art, including in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Such methods are generally described in Sambrook et al., (1989, 1992),in Ausubel et al., Current Protocols in Molecular Biology, John Wileyand Sons, Baltimore, Md. 1989.

A recombinant cell of the present invention comprises a cell transfectedwith a nucleic acid molecule that encodes a viral antigen of theinvention. A variety of expression vector/host systems may be utilizedto contain and express sequences encoding the viral antigens of theinvention. These include, but are not limited to, microorganisms such asbacteria transformed with recombinant bacteriophage, plasmid, or cosmidDNA expression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids);or animal cell systems. The invention is not limited by the host cellemployed. The expression of the construct according to the presentinvention within the host cell may be transient or it may be stablyintegrated in the genome thereof.

Vaccine Compositions and Methods Thereof

According to some aspects the present invention provides a vaccinecomprising an isolated viral antigenic peptide and a peptide comprisinga T cell epitope of HSP60, wherein the HSP60 peptide enhances theimmunogenicity of the viral antigenic peptide by at least two foldcompared to the peptide without the HSP60 peptide. In certain currentlypreferred embodiments the immunogenicity is enhanced by at least 4-5fold.

In certain embodiments the vaccine compositions comprise a T cellepitope of HSP60 suitable to enhance the immunogenicity when used as anadjuvant peptide that is mixed with the viral antigen. According tocertain particular embodiments, the adjuvant peptide is selected fromEc27 and analogs and derivatives thereof. In alternative embodiments thevaccine comprises a T cell epitope of HSP60 suitable to enhance theimmunogenicity of the viral antigenic peptide when used in conjugateswhere the HSP60 peptide is covalently linked to the viral antigenicpeptide. In some particular embodiments, the peptide carrier is selectedfrom p458, Ec27 and analogs and derivatives thereof. The enhancedimmunogenicity of said viral antigen is measured by at least one of thefollowing: serum titer of antibodies directed to said viral antigen; Tcell proliferation in the presence of said viral antigen; cytokinesecretion induced by said viral antigen; specific T cell mediated lysisof virus-infected cells; and reduction of detectable viral load.

In another aspect, the invention provides vaccine compositionscomprising the conjugates of the invention and a pharmaceuticallyacceptable carrier, adjuvant, excipient or diluent.

In another aspect, the invention provides vaccine compositionscomprising a polypeptide or peptide, said polypeptide or peptidecomprising an amino acid sequence as set forth in any one of SEQ IDNOS:11-12, 21 and 25-44, and a pharmaceutically acceptable carrier,adjuvant, excipient or diluent.

In one embodiment of the invention, the composition is useful fortreating or preventing a viral infection in a subject in need thereof,as described herein.

The vaccine composition of the invention is administered to a subject inneed thereof in an effective amount. According to the present invention,an “effective amount” is an amount that when administered to a subjectresults in a substantial increase in the immune response of the subjectto said viral antigen, as described herein.

According certain embodiments, the subject is selected from the groupconsisting of humans, non-human mammals and non-mammalian animals (e.g.birds). In a preferred embodiment, the subject is human.

Pharmaceutical and veterinary compositions for use in accordance withthese embodiments may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients (vehicles). Thecarrier(s) are “acceptable” in the sense of being compatible with theother ingredients of the composition and not deleterious to therecipient thereof. The vaccine composition can be optionallyadministered in a pharmaceutically or physiologically acceptablevehicle, such as physiological saline or ethanol polyols such asglycerol or propylene glycol.

The polypeptides and peptides of the invention may be formulated intothe vaccine as neutral or salt forms. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with free amino groups of thepeptide) and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids such as acetic,oxalic, tartaric and maleic. Salts formed with the free carboxyl groupsmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidineand procaine.

The vaccine composition may optionally comprise additional adjuvantssuch as vegetable oils or emulsions thereof, surface active substances,e.g., hexadecylamin, octadecyl amino acid esters, octadecylamine,lysolecithin, dimethyl-dioctadecylammonium bromide,N,N-dicoctadecyl-N′-N′bis(2-hydroxyethyl-propane diamine),methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran,dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide,dimethylglycine, tuftsin; immune stimulating complexes; oil emulsions(including, but not limited to, oil-in-water emulsions having oildroplets in the submicron range, such as those disclosed by U.S. Pat.Nos. 5,961,970, 4,073,943 and 4,168,308); liposaccharides such as MPL®and mineral gels. The antigens of this invention can also beincorporated into liposomes, cochleates, biodegradable polymers such aspoly-lactide, poly-glycolide and poly-lactide-co-glycolides, or ISCOMS(immunostimulating complexes), and supplementary active ingredients mayalso be employed. The protein and peptide antigens of the presentinvention can be coupled to albumin or to other carrier molecule inorder to modulate or enhance the immune response, all as are well knownto those of ordinary skill in the vaccine art.

The vaccines can be administered to a human or animal by a variety ofroutes, including but not limited to parenteral, intradermal,transdermal (such as by the use of slow release polymers),intramuscular, intraperitoneal, intravenous, subcutaneous, oral andintranasal routes of administration, according to protocols well knownin the art. The particular dosage of the conjugate antigen will dependupon the age, weight and medical condition of the subject to be treated,as well as on the identity of the antigen and the method ofadministration. Suitable doses will be readily determined by the skilledartisan. A preferred dose for human intramuscular, subcutaneous and oralvaccination is between about 6 μg to about 70 mg per kg body weight,preferably between about 15 μg to about 28 mg per kg body weight, andmore preferably between about 40 μg to about 7 mg per kg body weight.Adjustment and manipulation of established dosage ranges used withtraditional carrier antigens for adaptation to the present vaccine iswell within the ability of those skilled in the art.

In various embodiments, the vaccine composition s of the invention maybe used in combination with other treatments and medicaments, e.g.anti-viral drugs. For example, a conjugate comprising a CTL epitopederived from 1E-1 protein of HCMV and a peptide carrier of the inventionmay be administered to HCMV infected subjects in combination withgancyclovir therapy. Doses and administration regimes of gancyclovir areknown in the art.

In another aspect, the present invention is directed to the use of aconjugate of the invention for the preparation of a vaccine compositionuseful for conferring anti-vial immunity.

In yet another aspect, the invention provides methods for increasing theimmunogenicity of a viral antigen which comprises linking the antigen toa synthetic peptide carrier of the invention.

In another aspect, the invention provides methods for immunizing asubject in need thereof against a viral infection, comprisingadministering to the subject an effective amount of a vaccinecomposition comprising a conjugate of the invention and apharmaceutically acceptable carrier, adjuvant, excipient or diluent.

In various embodiments, the vaccine composition may be administered tosaid subject before the exposure of said subject to the virus or afterexposure of said subject to said virus.

In another aspect, the invention provides methods comprising:

-   -   (a) isolating a viral antigen, comprising at least one epitope        selected from: a CTL epitope, a B cell epitope and a MHC        II-restricted epitope;    -   (b) conjugating said viral antigen to a synthetic peptide        carrier of the invention; and    -   (c) administering to the subject an effective amount of a        vaccine composition comprising a conjugate of the invention and        a pharmaceutically acceptable carrier, adjuvant, excipient or        diluent.

Diagnostic Kits and Methods Thereof

Other embodiments of the present invention are directed to diagnosticcompositions and kits and uses thereof for the diagnosis of flavivirusinfection.

The present invention provides a method for diagnosing the presence of,or exposure to a flavivirus in a patient, comprising testing saidpatient for the presence of anti-flavivirus antibodies or of a T cellswhich immunoreact with flavivirus epitopes using a peptide according toTable 1 or analogs, derivatives and salts thereof as antigen.

In one embodiment, the method comprises the steps of:

-   -   (a) contacting a suitable biological specimen with a viral        antigen having an amino acid sequence as set forth in any one of        SEQ ID NOS:11-12, 21 and 25-44 and analogs, homologs,        derivatives and salts thereof under conditions such that an        immune reaction can occur;    -   (b) quantifying the immune reaction between the peptide antigen        and the biological specimen,    -   wherein an immune reaction significantly higher than an immune        reaction obtained for a sample obtained from a non-infected        subject is indicative of exposure to, or, in other embodiments,        infection of the subject with a flavivirus.

A biological specimen or sample that may be assayed for flavivirusinfection may include, for example, mammalian body fluids (e.g. serum,tissue extracts, tissue fluids, mucosal secretions), in vitro cellculture supernatants, cell lysates and cells or tissue from the subjectthat have been cultured in cell culture (e.g. leukocyte samples such asperipheral blood mononuclear cells). Methods of obtaining a suitablebiological sample from a subject are known to those skilled in the art.

In certain embodiments, the peptides and peptide compositions preparedin accordance with the present invention can be used to detectanti-flavivirus antibodies and diagnose flavivirus infection by usingthem as the test reagent in an enzyme-linked immunoadsorbent assay(ELISA), an enzyme immunodot assay, a passive hemagglutination assay(e.g., PHA test), an antibody-peptide-antibody sandwich assay, apeptide-antibody-peptide sandwich assay, or other well-knownimmunoassays. In accordance with the present invention, any suitableimmunoassay can be used with the subject peptides. Such techniques arewell known to the ordinarily skilled artisan and have been described inmany standard immunology manuals and texts. In one particularembodiment, the immunoassay is an ELISA using a solid phase coated withthe peptide compositions of the present invention. For example, such akit for determining the presence of anti-flavivirus antibodies maycontain a solid-phase immobilized peptide of the invention and a taggedantibody capable of recognizing the non-variable region of theanti-flavivirus antibody to be detected, such as tagged anti-human Fab.The kit may also contain directions for using the kit and containers tohold the materials of the kit. Any conventional tag or label may beused, such as a radioisotope, an enzyme, a chromophore or a fluorophore.A typical radioisotope is iodine-125 or sulfur-35. Typical enzymes forthis purpose include horseradish peroxidase, horseradish galactosidaseand alkaline phosphatase.

In other embodiments, the presence of T cells immunoreactive withflavivirus epitopes may be determined, for example, by determining Tcell proliferation or cytokine secretion induced by the novel viralpeptide epitopes of the invention, using methods well known in the art.Several non-limitative examples of determining T cell reactivity withpeptide antigens are presented in the Examples herein. For example, akit for diagnosing flavivirus exposure or infection by testing for thepresence of a T cell which immunoreacts with flaviviral epitopes, maycomprise: an antigen selected from the peptides of the invention; asuitable medium for culture of lymphocytes (T cells); and either alabeled nucleotide for the T cell proliferation test, or a cytokine,e.g., interferon-gamma, assay kit, for the cytokine test.

In various embodiments, the method may comprise the steps of:

-   -   (a) contacting a suitable biological sample with a viral antigen        having an amino acid sequence as set forth in any one of SEQ ID        NOS:11-12, 21 and 25-44 and analogs, homologs, derivatives and        salts thereof under conditions such that an immune reaction can        occur;    -   (b) determining whether the peptide antigen binds specifically        to the biological sample.

The term “binds specifically to the biological sample” as used hereinrefers to occurrence of an immune reaction between a component of thebiological specimen or sample (e.g. antibodies and T cells) and theviral peptide antigen having higher affinity or extent than to anotherantigen. For example, specific binding may be measured by determiningthe extent of antigen-antibody complex formation, T cell proliferationor cytokine secretion. Thus, for example, step (b) may includedetermining the extent of antigen-antibody complex formation, wherein anantigen-antibody complex formation level significantly higher than thelevel obtained for a sample obtained from a subject not previouslyexposed to or infected by a flavivirus is indicative of exposure of thesubject to the flavivirus.

The kits and methods of the present invention may be used, in someembodiments, for the differential diagnosis of a flavivirus infection,enabling the identification of the particular flavivirus straininfecting the subject or to which the subject was exposed. For example,a biological specimen may be assayed for the presence of anti-Dengueantibodies using the peptides having an amino acid sequence as set forthin SEQ ID NOS:30-33 to determine the strain of Dengue virus infectingthe subject (e.g. to distinguish between Dengue 1, 2, 3 or 4 infection).

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES A. CMV Vaccination

Materials and Methods

Mice

BALB/c female mice were purchased from Harlan Olac (Bicester, UK). Micewere maintained under specific pathogen free conditions and were allowedto adjust to the facility for 1 week before any experiments wereperformed. For the pathogenesis experiments, mice were used at 6 to 8weeks of age and for the immunization experiments, mice were used at 3weeks of age. The mouse experiments were approved by and performedaccording to the guidelines of the Ben Gurion University Faculty ofHealth Sciences Animal Safety Committee.

MCMV

The Smith strain of MCMV was obtained from the American Type CultureCollection (ATCC) (Rockville, Md.). Highly virulent salivarygland-passaged MCMV stocks were prepared as a 10% (wt/vol) homogenate ofsalivary gland from day 14-infected BALB/c in DMEM-10% FCS. Homogenateswere clarified by low speed centrifugation, DMSO was added to finalconcentration of 10%, and virus stocks were stored in aliquots at −70°C. until use (Palmon et al., 1996).

MCMV titers in these salivary gland suspension (SGS) stocks weredetermined by a quantitative plaque assay (Rager Zisman et al., 1973).Briefly, confluent monolayers of secondary mouse embryo fibroblasts(MEF) were prepared in 24 well plates. Serial 10-fold dilutions of SGScontaining MCMV were prepared in DMEM supplemented with 2% FCS. Thegrowth medium from each well in MEF plates was aspirated, and duplicatewells were inoculated with 0.2 ml of diluted SGS. After an adsorptionperiod of 1 hour at 37° C., monolayers were overlayed with 0.8 ml ofgrowth medium containing 0.75% carboxymethyl cellulose (CMC), incubatedfor 5 days at 37° C. in a humidified 5% CO₂ incubator, fixed in PBS-10%formaldehyde and stained with Crystal Violet to visualize virus plaques.Titers were expressed as log_(in) pfu/0.1 gr tissue. Thorough this studyvirus stocks containing 1.75×10⁸ pfu/0.1 g of tissue were used.

Infection with MCMV and Virus Titers in Target Organs

To study the course of MCMV infection in naïve or immunized BALB/c mice,mice were inoculated intraperitoneally (i.p.) with 5×10⁴ pfu of stockvirus in 0.2 ml PBS. Mice were sacrificed at different time points,spleens and salivary glands (pooled 3 mice per group at each time point)were removed and 10% (wt/vol) homogenates were prepared as previouslydescribed (Palmon et al., 1996). Samples were stored at −70° C. untilinfectious virus titrations were performed on primary cultures of MEF.

Preparation of DNA and Amplification by PCR

DNA was extracted from naïve and infected spleens and salivary glandusing QiAmp Tissue Kit (QIAGEN Inc. Chatsworth, Calif., USA), accordingto appropriate QiAmp protocols. DNA oligonucleotide primers weresynthesized according to the published sequence of MCMV gB gene (Rapp etal., 1992). The sequence of gB sense strand primer was based on the cDNAsequence no. 2416-2443 (5′-AAG-CAG-CAC-ATC-CGC-ACC-CTG-AGC-GCC-3′ SEQ IDNO:17) and the antisense no. 2745-2772(5′-CCA-GGC-GCT-CCC-GGC-GGC-CCG-CTC-TCG-3′ SEQ ID NO:18). This gB geneprimer pair amplifying a 356 bp segment was found the most sensitive inprevious studies (Palmon et al., 1996). For gene amplification, 1 ng ofDNA sample was added to the reaction mixture containing 200 nM eachdNTP, 100 pmol each primer, 1.0 mM MgSO₄, 10 mM KCI, 10 mM (NH₄)₂SO₄, 20mM Tris-HCl (pH 8.8), 0.1% Triton X-100 and 2 U of vent polymerase(Biolabs) in a total reaction volume of 50 n1 each. Samples wereamplified for 30 cycles in an automated thermal cycler (Perkin ElmerCetus, USA). Each cycle entailed denaturation at 94° C. for 60 sec,annealing at 68° C. for 90 sec and primer extension at 72° C. for 120sec. PCR products were electrophoretically separated on 1.5% agarosegel, stained with ethidium bromide, and photographed. The lower limit ofdetection for this method under the experimental conditions was 5femtograms of viral DNA corresponding to about 20 copies of the MCMVgenome (Palmon et al., 1996).

Peptides

Peptides were prepared in the Weizmann Institute of Science (Rehovot,Israel), and in Albert Einstein College of Medicine (New-York USA). Thepurity of the peptides was ascertained by analytical reversed-phase HPLCand amino acid (aa) analysis. The sequences of the six peptidessynthesized are: 89pep (MCMV pp89 ie1-CTL epitope, Reddehase et al.,1989)-YPHFHPTNL (SEQ ID NO:5); p458 (the active peptide derived frommouse HSP60, Konen Waisman et al., 1999)-NEDQKIGIEIIKRALKI (SEQ IDNO:2); p458-89pep (combined)-NEDQKIGIEIIKRALKIYPHFHPTNL (SEQ ID NO:6);negative control for p458 (the p431 peptide of the mycobacterial HSP60,442val-deleted)-EGDEATGANI-KVALEA (SEQ ID NO:7); control-89pep(combined)-EGDEATGANI-KVALEAYPHFHPTNL (SEQ ID NO:8); and TTp30-89pep(combined)-FNNFTVSFWLRVPKVSASHLEYPHFMPTNL (SEQ ID NO:9). The p30 of TT(aa 947-967) (Panina-Bordignon et al., 1989) (SEQ ID NO:10) is now beingused as a carrier peptide in various vaccines (Brander et al., 1996;Keitel et al., 1999). The mycobacterial p431 peptide (442val-deleted)was used as a negative control peptide since it is homologous insequence to mammalian p458, but did not elicit a CD4⁺-dependent immuneresponse against itself or p458.

Immunization and Challenge of Mice with MCMV

The immunizing dose of each peptide was equimolar to 15 μg of p458(Konen Waisman et al., 1999). All peptides were emulsified in incompleteFreund's adjuvant (IFA), and the volumes for intra-footpad (i.f.p.) andsubcutaneous (s.c.) injections were 50 μl and 100 μl respectively. Twodifferent protocols were used. To study the immune response of mice tothe chimeric peptide (p458-89pep), groups of 6-week old to 8-week oldmice were immunized once into the hind footpad with peptides emulsifiedin IFA. Ten days later several mice were sacrificed, and organs wereharvested for IFNγ and IL-4 assays. To study the protective efficacy ofthe combined peptide, groups of 3-week old mice were immunized andboosted according to the following protocol: mice were immunized i.f.p.on day (−24), and boosted s.c. two weeks later on day (−10). Ten dayslater (day 0), mice were challenged IP with 5×10⁴ pfu of MCMV. Mice weresacrificed on days 14, 21, and 28 after challenge, and organs wereharvested for virus titrations, PCR, cytotoxic T cell and cytokineassays.

Preparation of Spleen and Salivary Gland Mononuclear Cell Cultures

Spleen pulp was extruded from the capsule in a non-tissue culture Petridish in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100μg/ml streptomycin, 2 mM glutamine, 10 mM HEPES and 5% FCS (base-RPMI).Spleen cell suspensions were passaged through a cell strainer, washedonce, treated 2 min with ACK lysing buffer (0.15M NH₄Cl, 0.01 KHCO₃; 2ml/spleen) for elimination of erythrocytes, and washed twice inbase-medium. Splenocytes were resuspended in RPMI-1640 mediumsupplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mMglutamine, 10 mM HEPES, 5×10⁻⁵M β-mercaptoethanol and 10% FCS (completeRPMI) in a final concentration of 5×10⁶ cells/ml.

Salivary gland cell suspensions were prepared by initially cutting thesalivary glands into small fragments (<2 mm) in a non-tissue culturePetri dish. Fragments were treated with base-medium containing 1 mg ofcollagenase-dispase (Roche Diagnostics, Germany)/ml and 50 μg of DNase I(Boehringer Mannheim, Germany)/ml. After 1 h incubation in 37° C., cellswere resuspended in 45% Percoll (Sigma Chemical Co., Israel), overlayedon 66% Percoll and centrifuged at 800 g for 25 min. Mononuclear cellscollected at the interphase, were counted and resuspended incomplete-RPMI to a final concentration of 5×10⁶ cells/ml.

IFNγ and IL-4 ELISA Assays

Mononuclear cell cultures from spleens and salivary gland were preparedas described above. Cell suspensions were divided into 24 well plates(5×10⁶ cells/well) and were stimulated in vitro with either 10 μg/ml of89pep or p458 or with 5 μg/ml Concanavalin-A (Con-A). Cells wereincubated for 72 h (with or w/o stimulation) at 37° C. in a humidified5% CO2 incubator. After incubation supernatants were collected, and IFNγand IL-4 levels were measured using indirect ELISA according toPharmingen cytokine ELISA protocol (Pharmingen, San Diego, Calif.).

FACS Analysis of Cell Phenotypes and Intracellular IFNγ

For phenotypic analysis, spleen and salivary gland mononuclear cells ofMCMV-infected and naive mice were cultured as described above and werestained for CD8 and CD4, IFNγ and IL-5 using directly-labeled antibodies(PharMingen, San Diego, Calif.). Intracellular cell staining (ICCS) forIFNγ, IL-4 and IL-5 was performed using PharMingen's Cytofix/CytopermPlus kit with GolgiPlug (containing Brefeldin A) according to themanufacturer's instructions. Briefly, GolgiPlug was added to the 8h-incubated immune cell cultures (established as described above, withor w/o peptide stimulation). After the additional 6 h of incubation inthe incubator cells (minimum 10⁶ per sample) were harvested, washed inPBS supplemented with 2% FBS and 0.09% Sodium azide, and incubated in 50μl of FC blocker, labeled with anti-CD4 and anti-CD8 surface markers.Then cells were fixed, permeabilized and treated with anti-IFNγ,anti-IL-4 or anti-IL-5 antibodies for intracellular cytokine detection.Stained cells were immediately analyzed on a FACSCalibur flow cytometer(Becton-Dickinson, Mansfield, Mass.) and 50,000 to 100,000 events/samplewere acquired and analyzed with CellQuest software.

Cytotoxic T Cell Assay

The cytotoxic activity against the MCMV 89pep was assessed in a 4-hcytotoxic assay using the CytoTox 96 non-radioactive, colorimetric-basedkit (Promega, Madison, Wis.), according to manufacturer instructions.This assay is based on the quantitative measurement of lactatedehydrogenase, a stable cytosolic enzyme that is released upon celllysis. Spleen cell suspensions from immunized mice, prepared asdescribed above, were re-stimulated in vitro for 6 days with 89pep (10μg/ml) and rhIL-2 (25 IU/ml) from day 2. Target cells for the lysisassay were P815 cells (mastocytoma, H-2^(d)). P815 were eithernon-pulsed or pulsed with 89pep (1 μg/ml) for 2 h and then washed beforeincubation with effector cells. In all experiments shown, thespontaneous release was less than 25% of maximal release. Each point ina lysis assay represents the average of triplicate values. The range ofthe triplicates was within 5% of their mean.

Example 1 Natural History of MCMV Dissemination in Spleen and SalivaryGland

MCMV infection is characterized by different kinetics and viral loads indifferent organs (Mercer and Spector, 1986). BALB/c mice, 6-8 weeks old,were injected i.p. with 5×10⁴ pfu of MCMV. Mice were sacrificed on days1, 3, 7, 14 and 28 after infection, and spleens and salivary glands wereassayed for infectious virus and MCMV DNA. FIG. 1, shows a typicalpattern of MCMV replication in spleen (empty diamonds) and salivarygland (full squares). Virus replication peaked in the spleen on day 3after infection, and slowly declined thereafter (FIG. 1A). By day 14, noinfectious virus could be recovered from this organ. To detect MCMV DNAin infected organs, we used a sensitive PCR using a gB gene primer pairthat amplifies a 356 bp segment (Palmon et al., 1996). Viral DNA wasdetected in the spleen as early as day 1 after infection, peaked on day3 and by day 14 no DNA could be detected (FIG. 1B).

In the salivary gland, virus appeared on day 7. Virus replication inthis organ steadily increased, peaking by day 14 (3×10⁸ pfu/0.1 grtissue, FIG. 1A). A moderate decline in virus titers ensued, and at day28, 1.5×10⁶ pfu/0.1 gr tissue were still recoverable from the salivarygland. No infectious virus could be detected in the SG (and in any otherorgan) by day 42 post challenge (data not shown and Keitel et al., 1999)The detection of viral DNA was associated with the presence ofinfectious virus. DNA increased from day 7 to 14. Large amounts of viralDNA could still be detected on day 28 after infection (FIG. 1B). On thisbackground of viral dissemination, replication, splenic clearance andsalivary gland persistence, we evaluated the efficacy of immunizationwith the p458-89pep chimeric peptide. We also studied MCMV load in lungsafter challenge of naïve 6-8 week old mice; MCMV load (pfu) maximized onday 7 and disappeared by day 14 (data not shown). Thus, in our model weconcentrated on the salivary gland because it is considered as the majorsite for viral persistence of MCMV in mice (FIG. 1 and Ho, 1991;Koszinowski et al., 1990, Kirchner, 1983; Mercer and Spector, 1986).

Example 2 Immunization with p458-89Pep Suppresses MCMV Persistence inthe Salivary Gland

89pep is the H-2L^(d)-restricted YPHFMPTNL epitope of MCMV-pp89(Reddehase et al., 1989). We synthesized chimeric p458-89pep andcompared its protective efficacy against MCMV to that induced by 89pepalone or by negative control-89pep. p458 is a MHC class II-restrictedpeptide derived from murine HSP60 (aa 458-474) and capable of inducingCD4+T responses in BALB/c mice (Amir-Kroll et al., 2003). Themycobacterial HSP60 431-447 aa peptide (with a val deletion at position442) did not elicit an immune response to itself or to p458, and thusserved as a negative control peptide for immunization; control-89pep.

To investigate whether immunization with the different peptides woulddecrease MCMV replication in salivary glands, 3-week old BALB/c micewere immunized twice with IFA alone, 89pep, p458-89pep or control-89pep(FIG. 2). Three-week old, female BALB/c mice were immunized (i.f.p.)with various peptides, and were boosted (s.c.). Two weeks later, themice were challenged (i.p.) with 5×10⁴ pfu MCMV, day 0. Three mice fromeach group were sacrificed on days 14, 21, and 28 after challenge.

Peptides for vaccination were emulsified in IFA. Ten days after the lastimmunization, mice were challenged i.p. with 5×10⁴ pfu of MCMV (day 0).Mice were sacrificed on days 14, 21, and 28 after challenge, andinfectious virus titers and MCMV-DNA were measured by plaque and PCRassays in the salivary glands. As shown in FIG. 2B, no effect ofimmunization with any of the peptides could be demonstrated on days 14and 21 after virus challenge; on day 14, virus titers ranged from 8.1 to8.8 log₁₀ pfu/0.1 g, and on day 21 ranged from 7.1 to 8.0 log₁₀ pfu/0.1g.

On day 28, however, MCMV was not detectable in the salivary glands ofthe p458-89pep-immunized mice (<2 log₁₀ pfu/0.1 g). Immunization with89pep alone showed a marginal advantage compared to IFA-immunized mice(FIG. 2B); the viral load was 4.5 and 5.1 log₁₀ pfu/0.1 g, respectively.Immunization with control-89pep did not affect the viral load. Otherexperiments were performed with the same experimental design in whichTTp30-89pep was used; immunization with TTp30-89pep did not affect viralload on days 14 and 21, reduced viral load on day 28 by two fold, onaverage, but failed to eliminate infectious virus on day 28. To furtherevaluate the virus suppression induced by the p458-89pep immunization,we used a sensitive viral gB PCR to detect viral DNA. We previouslyshowed that 1 pfu is the equivalent of approximately 1500 viral genomes(Palmon et al., 2000). Yet, on day 28, even this assay failed to revealany gB PCR product in salivary glands of mice immunized with p458-89pep(FIG. 2C, lane d). Therefore, only immunization with the p458-89pep ledto the elimination of detectable MCMV from the salivary gland, on day28.

Example 3 IFNγ Secretion by 89Pep-Specific T Cells Following Infectionand Vaccination

It is well established that clearance of MCMV during acute infectiondepends primarily on Th1 IFNγ secretion and protective CTL responses(Mercer and Spector, 1986; Reddehase et al., 1989). We tested whetherIFNγ secretion was stimulated by 89pep from spleen (FIG. 3A) andsalivary gland (FIG. 3B) cell cultures of MCMV-challenged mice. Cellcultures were prepared on days 1, 3, 7, 14, 21, and 28 after infection,plated for 3 d with or without 89pep, and IFNγ secretion was measured.In the absence of 89pep stimulation, secretion of IFNγ was detected onlyin spleen cultures from days 1 and 3 after infection. This resultprobably reflects NK activity in the early stages of infection. When89pep was added to the cultures, IFNγ secretion in spleen and salivaryglands was correlated with the kinetics of viral replication in theseorgans (FIGS. 1A and 3). It is noteworthy that no significant IL-4secretion was detected in the culture supernatants; however, the cellswere capable of secreting IL-4 along with other cytokines in response tostimulation with Con-A (data not shown).

We investigated whether immunization with p458-89pep induced89pep-specific IFNγ secretion. Mice received a single immunization withthe following peptides: p458-89pep, 89pep, p458, control-89pep orTTp30-89pep. TTp30 is a MHC class II-restricted peptide capable ofinducing vigorous CD4+T responses and IFNγ production in BALB/c mice andused as a universal adjuvant (Panina-Bordignon et al., 1989, Amir-Krollet al., 2003). In addition, a non-vaccinated group was infected withMCMV. Ten days after immunization with the different peptides orinfection, mice were sacrificed, and spleen cell and salivary glandcultures were prepared and stimulated in vitro for 3 d with 89pep orp458. FIG. 4 shows that spleen cells derived from mice immunized withp458-89pep and re-stimulated in vitro with 89pep secreted significantlyhigher (p<0.05) levels of IFNγ compared to mice immunized with 89pep,p458, control-89pep or IFA-only. 89pep-restimulated splenocytes fromp458-89pep-immunzed mice secreted significantly higher (p<0.05) levelsof IFNγ compared to the same but non-re-stimulated splenocytes (FIG. 4).Thus, immunization with p458-89pep induced specific and significantlyenhanced IFNγ secretion. In these experiments we also tested theTTp30-89pep. Immunization with TTp30-89pep followed by 89pepre-stimulation induced IFNγ levels similar to those of mice immunizedwith p458-89pep (FIG. 4). The highest levels of 89pep-specific IFNγsecretion were obtained in spleen cell cultures from mice infected withvirus (FIG. 4). This high IFNγ secretion by spleen cells fromMCMV-infected mice, after in vitro stimulation with 89pep, indicates thedominance of this epitope in the response to MCMV. No 89pep-specificIFNγ was detected in salivary gland cell cultures after immunizationwith the different peptides (data not shown). Thus, infection of thesalivary gland with MCMV appeared to be needed for recruitment to theorgan of 89pep-specific IFNγ producing cells (FIGS. 1A and 3).

The response of spleen cell cultures to stimulation with p458 inducedhigh levels of IFNγ in mice immunized with p458 or p458-89pep, but notin other groups; this indicates that the responses were immunologicallyspecific (FIG. 4). No significant IL-4 secretion after eitherimmunization was detected; nonetheless the cells were capable ofsecreting IL-4 after stimulation with Con-A. IL-4 levels measured afterCon-A stimulation in vitro were 242 pg/ml, 146 pg/ml, 184 pg/ml and 317pg/ml for IFA-only, 89pep, p458, and p458-89pep respectively. Takentogether, these results imply that the protection induced by p458-89pepwas associated with elevation in MCMV-specific IFNγ production.

Example 4 Immunization with p458-89Pep Induces 89Pep-Specific IFNγ⁺CD8⁺T Cells and CTL Activity

We characterized the nature of cells secreting the IFNγ by flowcytometry. Mice were immunized once with the different peptides and anadditional group was infected with MCMV. Seven days later, spleens wereremoved and cell suspensions were cultured for 5 days with or without89pep. Immunization with p458-89pep followed by 5 days of re-stimulationwith 89pep induced IFNγ⁺CD8⁺ T cells and no IFNγ⁺CD4⁺ T cells (FIG. 5);very few IFNγ⁺CD8⁺ T cells were detected after immunization andre-stimulation with 89pep alone (FIG. 5B). Infection with MCMV andre-stimulation with 89pep induced the highest percentage of IFNγ⁺CD8⁺ Tcells (FIG. 5B). Staining was specific to IFNγ since no CD8⁺IL-4⁺ orCD8⁺IL-5⁺ cells were observed.

We also investigated whether the CD8⁺IFNγ⁺ cells induced by thep458-89pep were able to lyse specifically 89pep-loaded target cells.Mice were immunized once and 7 d later, spleens were harvested andre-stimulated with 89pep. Six days later, lytic activity was assayed onP815 (H-2^(d)) loaded with the 89pep. No lytic activity was observedfrom the cultures of 89pep-immunized mice, but CTLs induced byp458-89pep lysed the target cells (FIG. 6). Similar to our results withIFNγ production by CD8⁺ T cells, the 89pep-specific lytic activityinduced by MCMV infection was higher than that induced by p458-89pepimmunization.

Example 5 Salivary Gland-Specific Response after Immunization and VirusChallenge

We found, above, that IFNγ secretion in the salivary gland wasvirus-specific and depended on MCMV infection (FIGS. 1 and 3). In thepresent experiment, we monitored IFNγ production in immunized mice 28days after virus challenge. Staining of mononuclear cells forIFNγ-production was performed immediately after excision of the salivarygland, and stimulation with 89pep for 8 hr. The salivary glands of miceimmunized with IFA, 89pep or TTp30-89pep and challenged with viruscontained infectious virus on day 28 post challenge (FIG. 2). Likewise,CD8⁺IFNγ⁺ cells were observed in these day 28-infected salivary glands.In contrast, mice immunized with p458-89pep showed no infectious MCMV inthe salivary gland 28 days after infection (FIG. 2). Nevertheless, thenumber of CD8⁺IFNγ⁺ cells was larger than that of the other groups (FIG.7). This indicates that vaccination with p458-89pep induced a largereservoir of 89pep-specific CD8⁺ T cells along with termination ofsalivary gland infection.

Example 6 p458-89Pep Reduces Viral Load of MCMV-Infected Mice

3-week old BALB/c mice were challenged i.p. with 5×10⁴ pfu of MCMV (day0). On day 6, mice were immunized once with IFA alone, 89pep orp458-89pep emulsified in IFA, as described above. Mice were treated with100 μg of the anti viral medication Gancyclovir (GCV, Roche, Basel,Switzerland) i.p. on days 1, 2, 3, 4, 8, 9, 10 and 11 after challenge.Mice were sacrificed on day 30 after challenge and MCMV-DNA was measuredby a PCR assay in the salivary glands, as specified above (FIG. 14A).

As can be seen in FIG. 14B, immunization with p458-89pep was able tosuppress CMV load at the salivary gland even when applied after CMVchallenge and in combination with GCV. GCV treatment alone did notsuffice for therapy; also GCV treatment combined with one immunizationwith the non-conjugated 89pep did not affect CMV load. Only GCVtreatment combined with one p458-89pep immunization reduced viral loadto undetectable levels.

B. WNV Vaccination

Materials and Methods

Mice

BALB/c female mice were purchased from Harlan Olac (Jerusalem, Ill.) atthe age of 14 days (10-12 g body weight). Mice were maintained underspecific pathogen free conditions and were allowed to adjust to thefacility for 1 week before experiments were performed. Mice were used atthe age of 3-6 weeks unless otherwise stated. Age- and sex-matchedanimals were used as controls. Mice were maintained in isolation cagesand were fed and watered ad libitum. The mouse experiments were approvedand performed according to the guidelines of the Ben Gurion University,Faculty of Health Sciences, Animal Safety Committee.

Cell Cultures

The Vero cell line was derived from African Green Monkey (ATCC® number:CCL-81). The cells were grown in DMEM supplemented with 10% FCS, 1%nonessential amino acids and antibiotics. The cells were maintained in ahumidified atmosphere at 37° C. in 5% CO₂ and were used for growingvirus stocks, virus titration and neutralization assays.

Virus, Virus Titrations and WNV Antigen

The strain of West Nile Virus (WNV) was isolated from a human case ofWNV infection (Goldblum et al., 1954). Signature amino acid motifsindicate that this strain belongs to lineage I. Virus plaque assays wereperformed on Vero cell monolayers in 24 well plates as previouslydescribed (Ben-Nathan et al., 1996). Virus stock titers were expressedas plaque-forming units (pfu) per ml. A single virus stock containing5×10⁷ pfu/ml was prepared in Vero cells, stored in aliquots at −70° C.,and was used throughout this study. WNV antigen (WNV Ag) was prepared aspreviously described (Ben-Nathan et al., 2003).

Peptides

Peptides were synthesized at Sigma-Aldrich (Rehovot, Israel). Peptidepurity was ascertained by analytical reversed-phase HPLC and amino acid(aa) analysis and was assessed on >95% purity. The sequences of the sixpeptides synthesized are: Ep15 (derived from the E3 domain ofWNV)-LVTVNPFVSVATANS, SEQ ID NO:11; p458 (the active peptide derivedfrom mouse HSP60)-NEDQKIGIEIIKRALKI, SEQ ID NO:2; p32 (p458-Ep15combined)-NEDQKIGIEIIKRALKILVTVNPFVSVATANS, SEQ ID NO:13; pmock, anegative control for p458 (the p431 peptide of the mycobacterial HSP60,442val-deleted)-EGDEATGANIKVALEA, SEQ ID NO:7; p458-89pep (combined p458and 89pep, 89pep is a nonapeptide, YPHFHPTNL SEQ ID NO:5, which consistsof a MCMV pp89 ie1-CTL epitope)-NEDQKIGIEIIKRALKIYPHFHPTNL, SEQ ID NO:6.The mycobacterial p431 peptide (442val-deleted) was used as a negativecontrol peptide since it is homologous in sequence to mammalian p458,but did not elicit a CD4⁺-dependent immune response or antibodiesagainst itself or p458.

Antibodies and Sera

Human intravenous immunoglobulin-IL (IVIG-IL): the IgG preparation fromIsraeli donors (IVIG-IL; OMRIGAM 5% intravenous IgG) containing 50 mg/mlIgG (total protein 5% w/v) was a gift from Omrix Biopharmaceuticals Ltd,Israel. This product has an anti-WNV antibody titer of 1:1600 by ELISAand of >1:80 by plaque-reduction neutralization testing (PRNT)(Ben-Nathan et al., 2003). Mouse WNV antiserum was prepared byintraperitoneal (IP) injection of 5-week old BALB/c mice with 1×10⁴ pfuof WNV per mouse. Two weeks later, surviving mice were boosted with1×10⁴ pfu and bled 7 days later. Blood was centrifuged (4000 rpm for 7min), and serum was collected and stored at −20° C. The antibody titer,measured by ELISA, was 1:2400. Serum from mock-injected naïve mice wasused as a negative control.

Recognition of WNV-Ag and Peptides by IVIG and Mouse Sera

ELISA tests were performed according to the method described by Martinet al (Martin et al, 2000) with slight modifications. Briefly,microtiter plates were coated and incubated overnight at 4° C. with 100μl of the different peptides (1 μg/well) or WNV antigen diluted 1:700 incoating buffer (NaHCO₃, pH=9.6). After incubation, the coating bufferwas decanted and the plates were washed twice in PBS containing 0.05%Tween 20 and 0.2% sodium azide (washing buffer). After blocking for 1 hwith a 200 μl/well of PBS containing 0.05% Tween 20 and 2.5% nonfat drymilk, the plates were washed 4 times in washing buffer and 100 μl ofIVIG-IL or mouse sera at 1:40 dilution were added to each well (2-4wells per sample). Negative and positive controls of human or mouse serawere tested in each plate. After incubation for 1 h at 37° C. in ahumidified atmosphere, the plates were washed 5 times, and 100 μl of1:1000 diluted HRP-Streptavidin-conjugated anti-human IgG(Sigma-Aldrich) or 1:1000 diluted HRP-Streptavidin-conjugated anti-mouseIgG (SouthernBiotech, Birmingham, Ala.) respectively, was added to eachwell. After incubation at 37° C. for 1 h, the plates were washed 5 timesand 100 μl of TMB substrate (DAKO Carpinteria, Calif.) was added to eachwell and incubated at room temperature for 30 min. The color intensitywas measured by ELISA-reader (Dynatec MR 5000) at the absorbance of 405nm.

Lymphocyte Proliferation Assay

Splenocytes from immunized or naive mice were prepared as describedabove, resuspended in complete RPMI in a final concentration of 2×10⁵cells per well in 96-well plates and stimulated in vitro with either 10μg/ml of different peptides, 10 μl/ml of WNV-Ag, or 5 μg/ml ofConcanavalin-A (Con-A). Cell cultures were incubated at 37° C. in 5% CO₂for 5 days. At the end of incubation, 1 μCi of ³H-Thymidine (AmershamBiosciences, Buckinghamshire, England) was added to each well for 12 h.Radioactive counting was performed on a β-counter (WALLAC 1409).

Immunization of Mice with Peptides and WNV Challenge

The immunizing dose of each peptide was equimolar to 15 μg of p458(Konen Waisman et al., 1999). All peptides were emulsified in incompleteFreund's adjuvant (IFA), and injected at 50 μl/mouse intrafootpad (IFP).Mice were immunized with the different peptides 2-3 times according tothe experimental protocol. One week after the last immunization, micewere bled and sacrificed. Spleens were harvested, splenocyte culturesprepared and tested for T cell proliferation and cytokine secretion.Blood samples were centrifuged (4000 rpm for 7 min), and then sera werecollected and tested for anti-WNV antibodies by ELISA and neutralizationassays.

To study the immunogenicity and protective efficacy of the peptides,groups of 3-week old mice were immunized and boosted according to thefollowing protocol: mice were immunized IFP and boosted once or twice, 1week apart. One week after the last boost, the mice were challenged IPwith 1×10⁶ pfu of WNV. Mortality was recorded for the next 21 days. Forvirology studies, surviving and moribund mice were sacrificed on day 7after the challenge, and organs were harvested for virus titrations andRT-PCR.

Virus Load in Brain Tissue of Infected Mice

Brain tissues were removed from infected or immunized and challengedmice, and 10% (wt/vol) homogenates were prepared in DMEM-10% DMSO. Thehomogenates were then aliquoted and stored at −70° C. until furtheranalysis. Virus levels were determined by plaque titration on Vero cellmonolayers as previously described (Ben-Nathan et al., 1996), andexpressed as pfu/0.1 gr brain tissue.

RNA Extraction and RT-PCR

RNA was extracted from brain tissues from mice using RNEasy Midi Kit(QIAGEN, Hilden, Germany), and RT-PCR was performed on RNA extractsusing Endo-Free Reverse Transcriptase (Ambion, Huntingdon, UK) andBiomix-Red (Bioline, London, UK). WNV E protein primers(5′-ACGAAGTGGCCATTTTTGTC-3′, SEQ ID NO:81/5′-TTGATGCAGAGCTCCCTCTT-3′,SEQ ID NO:82) were chosen using Primer3 program (Whitehead Institute forBiomedical Research). PCR products were electrophoretically separated on1.5% agarose gel, stained with ethidium bromide and imaged using a CCDcamera (Imagechem 5500).

Preparation of Spleen Cell Cultures and Cytokine Analysis

Spleens of immunized mice were harvested, and spleen pulp was extrudedfrom the capsule in RPMI-1640 medium supplemented with 100 U/mlpenicillin, 100 μg/ml streptomycin, 2 mM glutamine, 10 mM HEPES and 5%FCS (base-RPMI). Spleen cell suspensions were passed through a cellstrainer, washed, treated for 2 min with ACK lysing buffer (0.15M NH₄Cl,0.01 KHCO₃) for elimination of erythrocytes, and washed twice inbase-medium. Splenocytes were resuspended in RPMI-1640 mediumsupplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mMglutamine, 10 mM HEPES, 5×10⁻⁵M β-mercaptoethanol and 10% FCS (completeRPMI) in a final concentration of 5×10⁶ cells/ml in a 24-well plates.Cell suspensions were stimulated in vitro with either 10 μg/ml ofdifferent peptides, 10 μl/ml of WNV-Ag, or 5 μg/ml of Con-A. The cellswere then incubated at 37° C. in 5% CO₂ for 72 h. Supernatants werecollected, diluted by 2, and IFNγ and IL-4 concentrations were measuredby indirect ELISA method using commercial kits (PharMingen, San Diego,Calif.) according to manufacturer instructions.

Virus Plaque Reduction Neutralization Testing (PRNT)

The titer of neutralizing antibodies was determined using a modifiedplaque reduction test (PRNT). Briefly, serial 2 fold dilutions (1:4 to1:512) of mouse sera were prepared in 96-well flat-bottom microtiterplates and 10⁴ pfu of WNV in equal volumes was added to duplicate wellsof each dilution. After 30 min of incubation at room temperature, 5×10⁴of Vero cells were added to each well of the sera-virus mixtures. Theplates were incubated for 72 h in a humidified atmosphere at 37° C. and5% CO₂ and plaques were counted. Plaque-reduction neutralizing antibodytiters were expressed as the reciprocal of the highest dilution thatgave 50% plaque reduction (PRNT₅₀).

Isotypes of Anti-WNV Antibodies

Plates were coated with the WNV-Ag diluted 1:700 in coating buffer.After overnight incubation at 4° C., blocking and washing, variousdilutions of mouse sera were added in triplicates. Negative and positivecontrols of mouse sera were tested in each plate. Total IgG assessmentwas performed using HRP-Streptavidin-conjugated anti-mouse IgG(SouthernBiotech). For antibody isotype identification, incubation withbiotin-conjugated anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgMantibodies (PharMingen, San Diego, Calif.) was followed by incubationwith 1:1000 diluted HRP-Streptavidin (Jackson Laboratories, West Grove,Pa.). After substrate addition the color intensity was measured byELISA-reader at 405 nm.

Example 7 Identification of a WNV Epitope

Based on the antigenic propensity method (Kolaskar et al., 1990),calculated using a free B-cell epitope program (BcePred Server,http://www.imtech.res.in/raghava/bcepred/index.html), and based on afree MHC epitope program ProPred server,http://www.imtech.res.in/raghava/propred), we have identified a p15peptide from different WNV-E proteins (from the immunogenic E3 domain,aa LVTVNPFVSVATANS or aa LVTVNPFVSVATANA; SEQ ID NOS:11 and 12,respectively) as a candidate B-cell continuous epitope andMHC-II-restricted epitope of the WNV (recognized by different human andmurine MHC-II molecules).

Next, it was examined whether the candidate epitope is recognized by aserum immunoglobulin pool taken from Israeli donors (WIG-IL), whichcontains anti-WNV Abs. Wells were coated with the different peptides (1μg/well) or with the WNV-Ag (1:700 dilution). After blocking andwashing, IVIG-IL was added at 1:40 dilution and binding was detected.The results demonstrate for the first time that the p15 peptide isrecognized by IVIG-IL (FIG. 8). Subsequently, mice were challenged IPwith a sublethal dose of WNV (10⁴ pfu) WNV and 14 days later survivingmice were re-infected with the same dose of WNV. Sera were collected 7days after the second challenge, and ELISA assays were performed. As canbe seen in FIG. 9, serum from WNV-infected mice specifically recognizedp15, and, to a greater extent, the p15 conjugate p32. In addition, Tcells from these mice proliferated in response to in vitro stimulationwith the p15 conjugate p32 (FIG. 10). Thus, the phenotype of p15 asWNV-B-cell epitope and WNV-MHC II-restricted peptide was herebydemonstrated.

Example 8 Immunization with p458-p15 Elicits WNV Protection

We showed that HSP60-p458 (NEDQKIGIEIIKRALKI SEQ ID NO:2) used as acarrier peptide for a MHC-I epitope of CMV enhanced immune response toCMV and immunization with the chimeric peptide, p458-CMV epitope,cleared MCMV from salivary glands of mice challenged with the virus. Inaccordance we tested the WNV-protective efficacy of p458-p15(NEDQKIGIEIIKRALKILVTVNPFVSVATANS) chimeric peptide (termed as p32, SEQID NO:13). Table 2 summarizes 5 different experiments; immunization withp15 alone showed slight protective efficacy while immunization with amixture of p15 and p458 did not protect mice. Nonetheless, when micewere immunized with p32, the chimeric p458-p15 peptide, a high degree ofprotection against a fatal challenge with virulent WNV was achieved.

TABLE 2 In vivo protective efficacy of p32 immunization against WNVchallenge Treatment - Deaths/Total (% deaths) No p458 p15 + treatmentp15 (x3) p32 (x3) (x3) p458 (x3) Mortality 22/32 8/15 2/21 4/7 5/7 (deadof total) (69%) (53%) (10%) (57%) (71%)

Mice were immunized 3 times with the different peptides or a peptide mixat 7 day intervals. Seven days after the 3rd immunization mice werechallenged with 10⁶ pfu of WNV and survival was monitored for 21 dayspost challenge. Results are the summary of 5 different experiments,performed in the same conditions.

To investigate whether immunization with p32 resulted in clearance ofWNV, virus levels in the brain, which is the prime target organ of thisneurotropic virus, were examined.

Mice were immunized 3 times with p32 at 7 day intervals. Seven daysafter the last immunization, immunized and non-immunized mice werechallenged IP with 10⁶ pfu of WNV. Seven days after challenge, mice weresacrifice and different organs were extracted (brain, lungs, heart,liver and spleen). Organs were taken also from control naïve mice(non-immunized and non-infected matched mice). Viral loads weredetermined by RT-PCR for viral genome and plaque assay for infectiousvirus titers (pfu/0.1 gr tissue). RT-PCR results are representative fromone experimental mouse while infectious titers are the average for allexperimental groups. * Detection level of the plaque assay is <10¹.

Virus levels were determined in the brain by RT-PCR and plaque assay onday 7 after challenge (FIG. 15). Virus titers were up to 4×108 pfu/ml inthe non-immunized and challenged mice, while in the naïve and in theimmunized and challenged group no virus was detected (FIG. 15). Thesefindings confirm that vaccination of mice with the p32 peptide protectsthem against an otherwise fatal WNV infection.

Example 9 Immunization with p458-p15 Induces WNV-Neutralizing Abs

We next investigated the immune responses elicited followingimmunization with p32. Immunization with p32 induced high titer ofWNV-specific Abs as compared to immunization with p15 alone (FIG. 11A).The Ab generated following immunization with p32 exhibitedWNV-neutralizing capacity, essential for the protective effect of thep32 vaccine (Table 3).

TABLE 3 Titers of anti-WNV neutralizing Abs in the sera of p32-immunizedor WNV-infected mice on day 7 after treatment Source of serum Titers ofanti-WNV neutralizing Abs Naïve mice <1:10   p32-immunized mice 1:80WNV-infected mice  1:320

Mice were either immunized 3 times with p32 or challenged with WNV.Seven days after the 3rd immunization or WNV challenge, respectively,mice were bled and titers of WNV-neutralizing Abs in sera were tested(PRNT₅₀). Results are the average of 4 independent ELISA experiments.

Next, it was examined whether p32 immunization and sublethal infectionwith WNV induce similar isotypes of anti-WNV antibodies. WNV-Ag was usedas the antigen in the ELISA plates. FIG. 1B shows that levels ofanti-WNV IgG1 and IgG2a isotypes were similarly high in sera fromp32-immunized and WNV-infected mice. WNV-specific IgG2b levels werehigher in sera from WNV-infected mice while WNV-specific IgM were higherin p32-immunized mice. Neither IgA nor IgG3 specific for WNV weredetected in both types of sera (FIG. 11B).

Example 10 Immunization with p458-p15 Induces WNV-Specific T CellResponse and IFN-γ Secretion

WNV-specific T-cell responses were examined as follows: Six-week oldBALB/c mice were infected with a sublethal dose of WNV (0.66×10² pfu),and spleens were harvested 6 days later. Spleen cell suspensions wereprepared, cultured in 96 wells plates and incubated for 3 days with the10 μg/ml of EP15, p32, p458 or Con-A (5 μg/ml) for 3 days. Cellproliferation was measured by the WST-1 method and the results are theaverage of 3 independent experiments. As shown in FIG. 12, splenocytesfrom mice immunized with p32 proliferated in response to in vitrostimulation with WNV antigen. This stimulation also induced secretion ofhigh levels of IFNγ, essential for TH1 response that characterizesvirus-protective immune responses (FIG. 13).

Example 11 Immunization with Conjugates of p458 and Ec27 with ViralAntigens

Conjugates of p458 and Ec27 with the novel viral antigens of theinvention, having amino acid sequences as set forth in SEQ ID NOS:13-14,23, 56-75 and 77-79 (see Table 4 below), are synthesized as describedabove.

Ec27 conjugate Virus P458 conjugate (SEQ ID NO): (SEQ ID NO): WNVNEDQKIGIEIIKRALKILVTVNPFVSVATANS (13) KKARVEDALHATRAAVEEGVGRLVNEDQKIGIEIIKRALKILVTVNPFVSVATANA (14) TVNPFISTGGANNKVM (77)NEDQKIGIEIIKRALKIYIVVGRGEQQINHHWHK (23) KKARVEDALHATRAAVEEGVLVTVNEDQKIGIEIIKRALKIGRLVTVNPFVSVATANS (65) NPFVSVATANA (78)NEDQKIGIEIIKRALKIGRLVTVNPFVSVATANA (66) KKARVEDALHATRAAVEEGVYIVVGRGEQQINHHWHK (79) Yellow NEDQKIGIEIIKRALKIGILVTVNPIASTNDDEVLIE (56)KKARVEDALHATRAAVEEGVGILV fever virus TVNPIASTNDDEVLIE (67) St. LouisNEDQKIGIEIIKRALKIGRLVTVNPFISTGGANNKVM (57) KKARVEDALHATRAAVEEGVGRLVencephalitis virus TVNPFISTGGANNKVM (68) Murray ValleyNEDQKIGIEIIKRALKIGRMVTANPYVASSTANAKVL (58) KKARVEDALHATRAAVEEGVGRMVencephalitis virus TANPYVASSTANAKVL (69) Kunjin virusNEDQKIGIEIIKRALKIGRLVTVNPFVSVSTANAKVL (59) KKARVEDALHATRAAVEEGVGRLVTVNPFVSVSTANAKVL (70) JapaneseNEDQKIGIEIIKRALKIGRLVTVNPFVATSSANSKVL (60) KKARVEDALHATRAAVEEGVGRLVencephalitis virus TVNPFVATSSANSKVL (71) DengueNEDQKIGIEIIKRALKIGRLITANPIVTDKEKPVNIE (61) KKARVEDALHATRAAVEEGVGRLIvirus type 1 TANPIVTDKEKPVNIE (72) DengueNEDQKIGIEIIKRALKIGRLITVNPIVTEKDSPVNIE (62) KKARVEDALHATRAAVEEGVGRLIvirus type 2 TVNPIVTEKDSPVNIE (73) DengueNEDQKIGIEIIKRALKIGRLITANPVVTKKEEPVNIE (63) KKARVEDALHATRAAVEEGVGRLIvirus type 3 TANPVVTKKEEPVNIE (74) DengueNEDQKIGIEIIKRALKIGRIISSTPLAENTNSVTNIE (64) KKARVEDALHATRAAVEEGVGRIIvirus type 4 SSTPLAENTNSVTNIE (75)

Mice are immunized 3 times with the different peptides or with controlpeptides (corresponding non-conjugated viral antigens and with thecarrier peptides alone) at 7 day intervals, as described above. Sevendays after the 3rd immunization mice are challenged with 10⁶ pfu of thecorresponding flavivirus and survival is monitored for 21 days postchallenge.

In other experiments, the immunogenicity of the conjugates is determinedby the following assays: in some experiments, mice are immunized andassayed for serum titers of virus-specific and viral peptide-specificantibodies by ELISA essays, as described above. In other experiments,LNC from immunized mice are examined in the presence of thecorresponding viral antigen in proliferation assays and IFN-γ secretionassays, as described above.

Example 12 Use of Ec27-Antigen Conjugates for Increasing theImmunogenicity of a Viral Antigen

Mice were immunized with the following peptides as described in Example8 using the following peptides: p15 (SEQ ID NO:11), p32 (SEQ ID NO:13),Ec27-p15 (KKARVEDALHATRAAVEEGVLVTVNPFVSVATANS, SEQ ID NO:77), andMock-p15 (EGDEATGANIKVALEALVTVNPFVSVATANS, SEQ ID NO:80—the p431 peptidefused to p15).

Spleens were harvested 10 days after the immunization and splenocyteswere cultured with p15 (25 μg/ml) for 3 days. Cell proliferation andIFNγ levels in the supernatants of spleen cell cultures were measured.Results are the average of 3 independent proliferation experiments.Ec27-Ep15* and Ec27-Ep15** are two identical but independent groups inthe same experiment.

As shown in FIG. 16, p458 and Ec27 were both able to increase theimmunogenicity of p15. Vaccination with conjugates comprising eitherp458 or Ec27 fused to the N-terminus of p15 resulted in enhancedproliferation and IFN-γ secretion of spleen cells in the presence ofp15, compared to those of spleen cells derived from animals vaccinatedwith p15 conjugated to the control peptide. Spleen cells from miceimmunized by IFA alone or unconjugated Ec27 did not induce significantproliferation and IFN-γ secretion when incubated with p15.

Example 13 Specific Recognition of the WNV Peptides by Sera Derived fromWNV-Exposed Subjects

Sera from WNV-exposed and non-exposed human subjects was assayed forantibodies recognizing the novel WNV peptide antigens, p15 (SEQ IDNO:11) and p17 (SEQ ID NO:21). FIG. 17 presents the results of an ELISAtest in which the p15 and p17 served as the antigen, human sera wereused as the primary antibody and HRP-conjugated anti-human Abs were usedas the secondary antibody. RLU are the relative luminescence unitsproduced by HRP activity on the luminal substrate.

Sera S2 and S6 are from human that were not infected with WNV while seraS1, S3-S5 and S7 are from human that had history of WNV infection. IVIGis pool of plasmas from Israeli citizens that was shown to containantibodies against WNV.

As can be seen, both p15 and p17 were specifically recognized byWNV-exposed sera and not by sera from unexposed subjects. Human serafrom Dengue-infected humans also did not recognize the p15 (data notshown). Thus, p15 and p17 are suitable diagnostic peptides useful fordetermining WNV exposure or infection.

Example 14 Identification of Ec27, an Immunodominant Peptide Derivedfrom E. coli GroEL

To find dominant T helper cell epitopes derived from the sequence of theE. coli variant of HSP60 (GroEL), BALB/c mice were inoculated withheat-inactivated E. coli bacteria and the proliferative responses ofdraining LNCs to a set of overlapping GroEL peptides (Table 5), and thewhole GroEL molecule (Purchased from Sigma), were analyzed.

TABLE 5 Overlapping peptides of the E. coli HSP60 molecule (GroEL);amino acid designation is corresponding to accession number gi: 45686198without the first methionine residue, SEQ ID NO: 83. Peptide NumberPosition  1  1-20  2 16-35  3 31-50  4 46-65  5 61-80  6 76-95  7 91-110  8 106-125  9 121-140 10 136-155 11 151-170 12 166-185 13181-200 14 196-215 15 211-230 16 226-245 17 241-260 18 256-275 19271-290 20 286-305 21 301-320 22 316-335 23 331-350 24 346-365 25361-380 26 376-395 27 (Ec27) 391-410 28 406-425 29 421-440 30 436-455 31451-470 32 466-485 33 481-500 34 496-515 35 511-530 36 526-545 37526-547

BALB/c mice were immunized s.c. with 10⁷ glutaraldehyde-attenuated E.coli bacteria (FIG. 18A) in PBS, or with 30 μg GroEL in PBS (FIG. 18B).Ten days later lymph node cells (2×10⁵ cells per well) were assessed forspecific proliferation to the indicated overlapping GroEL peptides (20μg/ml). After 96 hours of incubation, the ³H-thymidine incorporation wasassessed as a measure of proliferation. Results are shown as mean cpm ofquadruplicate wells. The standard deviations are indicated.

T-cell proliferation assays were performed as follows: Draining lymphnode cells (LNC) of immunized mice were cultured (2×10⁵/well) in 200 μlRPMI 1640 medium supplemented with 2 mM glutamine, non-essential aminoacids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin(BioLab, Jerusalem, Israel), 5×10⁻⁵M β-mercaptoethanol (Fluka AG, Buchs,Switzerland), 10 mM HEPES buffer (Sigma), and 1% syngeneic normal mouseserum. After four days of incubation, [³H]-thymidine (0.5 μCi of 5Ci/mmol, Nuclear Research Center, Negev, Israel) was added foradditional sixteen hours, and the thymidine incorporation was measured.Results are expressed as the mean cpm, or the stimulation index (SI),i.e. the mean cpm of test cultures (with antigen) divided by the meancpm of control cultures (without antigen).

As can be seen in FIG. 18A, a peptide corresponding to amino acidresidues 391-410 of GroEL was highly immunogenic. Immunization of BALB/cmice with the GroEL molecule instead of the whole E. coli bacteria ledto a similar proliferative response of draining LNC (FIG. 18B) to thesame GroEL peptides and the whole GroEL molecule as used for FIG. 18A:both immunogens gave rise to an immune response to the GroEL moleculeand, predominantly, to the Ec27 peptide of GroEL.

It was also tested whether the Ec27 peptide itself is immunogenic byimmunization of BALB/c mice with 20 μg of the Ec27 peptide in IFA.

BALB/c mice were immunized s.c. with 20 μg Ec27 peptide emulsified inIFA. Ten days later lymph node cells were taken and assessed forspecific proliferation of 2×10⁵ cells in the presence of the Ec27peptide, the acetylcholine receptor peptide 259-271 (AcR 259-271,VIVELIPSTSSAV SEQ ID NO:84), or GroEL at the concentrations 10 μg/ml, 2μg/ml, or without antigen (BG). Results are shown as mean cpm ofquadruplicate wells. The standard deviations are indicated.

FIG. 19 shows that the lymph node cells of immunized mice responded theEc27 peptide and to the whole GroEL molecule, but not to the controlpeptide AcR 259-271.

To learn whether the immunogenicity of the Ec27 peptide is restricted toa certain MHC haplotype, we compared four strains mice with differentMHC haplotypes in their response to the Ec27 peptide. The immunized micewere of the BALB/c (H-2^(d)), BALB/k (H-2^(k)), BALB/b (H-2^(b)), or SJL(H-2^(s)) strain. As can be seen in FIG. 20, all four different mousestrains responded specifically to the Ec27 immunogen peptide. Thus, theEc27 peptide could replace the GroEL, or the whole bacteria in primingfor a GroEL-specific immune response, due to its immuno-dominance.

Example 15 Use of the Ec27 Peptide as an Adjuvant for Antibody Responses

Because the Ec27 peptide was found to be an immunodominant peptide ofthe GroEL molecule, which is the major immunogen of bacteria, it wasinteresting to learn whether the Ec27 peptide can be used as anadjuvant, as can the bacteria. Therefore, BALB/c and C57BL/6 mice wereimmunized subcutaneously with 20 μg of the monoclonal anti-p53 antibodyPAb-246 (described in Yewdell et al., 1986) in different immunogeniccompositions: the antibody was either emulsified in Complete Freund'sAdjuvant (246 in CFA), in Incomplete Freund's Adjuvant (246 in IFA), ortogether with 50 μg the Ec27 peptide (as a mixture) in IFA (246 inEc27). Three weeks later, all mice received a boost of the PAb-246antibody subcutaneously in IFA. Ten days after the boost, mice were bledand their antibody responses to the PAb-246 immunogen were compared byELISA (FIG. 21A). Immunization of the PAb-246 antibody in IFA alone didnot result in a significant antibody response. In contrast, immunizationwith the antibody in IFA mixed with the Ec27 peptide resulted in aneffective antibody response in both strains. The adjuvant effect of theEc27 peptide was comparable to the effect of CFA, which is one of themost potent adjuvant materials known. FIG. 21B shows the induction ofanti-p53 antibodies in the immunized mice by ways of an idiotypicnetwork, i.e. these anti-p53 antibodies are anti-idiotypic to theanti-PAb-246 antibodies. Again, the use of the Ec27 peptide as anadjuvant was at least as effective as the use of CFA.

Dots represent individual sera, bars indicate the median of each group.

Example 16 Use of the Ec27 Peptide as an Adjuvant forp458-Polysacharride Conjugates

S. pneumoniae serotype 4 capsular polysaccharide (PnTy4) was obtainedfrom the American Type Culture Collection (ATCC; Rockville, Md., USA).PnTy4 was coupled to carrier peptides by using1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CDI;Aldrich, Wis., USA) using standard procedure.

The peptide carriers used in this example are: p458 (SEQ ID NO:2), p458s(SEQ ID NO:87, EGDIETGVNIVLKALTA), MOG (SEQ ID NO: 85,MEVGWYRSPFSRVVHLYRNGK).

BALB/c mice were immunized with the indicated peptide-polysaccharideconjugate in IFA or IFA mixed with 50 μg Ec27 as described above, in theWNV vaccination example. LNC were collected and assayed for nitric oxide(NO) production and proliferation.

Table 6 shows the levels of Nitric Oxide (NO) Production (nM) in lymphnode cells (LNC) derived from mice immunized with peptide-sugarconjugates in IFA or in IFA+Ec27. The cells were cultured for 72 h witheither Concanavalin A (Con A) or the peptide-sugar immunogen in thepresence or absence of a macrophage cell line (J774). NO production wasassayed using a Nitrate/Nitrile colorimetric assay according to themanufacturer's instructions.

LNC LNC + J774 Immunogen BG Con A P458-Ty4 BG Con A P458-Ty4P458-Ty4/IFA 0.13 4.73 0.21 0.55 6.24 10.29 P458-Ty4/ec27 0.18 3.32 2.360.66 4.33 34.97 LNC LNC + J774 P458s- P458s- Immunogen BG Con A Ty4 BGCon A Ty4 P458_(S)-Ty4/IFA 0.13 9.91 0.55 0.93 6.35 0.66 P458_(S)- 0.188.53 2.41 0.45 5.23 27.73 Ty4/ec27

The proliferation of lymph node cells in response to immunogen wasperformed as described above (Example 14).

Table 7 shows the proliferation of LNC derived from mice immunized withpeptide-sugar conjugates in IFA or in IFA+ec27. The cells were culturedfor 72 h with Concanavalin A (Con A), the peptide-sugar immunogen (Ty4),the peptide immunogen without the sugar, or the sugar conjugated to acontrol peptide (MOG-Ty4). The proliferative response is given as thestimulation index (SI).

TABLE 7 proliferation of LNC derived from mice immunized withpeptide-sugar conjugates in IFA or in IFA + ec27. Immunogen BG Con AP458-Ty4 P458 MOG-Ty4 P458-Ty4/IFA 1.0 2.3 4.7 2.9 2.7 P458-Ty4/ec27 1.06.5 15.4 3.1 4.0 Immunogen BG Con A P458_(S)-Ty4 P458_(S) MOG-Ty4P458_(S)-Ty4/IFA 1.0 12.8 2.9 2.2 1.3 P458_(S)-Ty4/ec27 1.0 15.3 8.7 2.16.5

In the tables, BG refers to background values; ConA refers toconcanavalin A; P458-Ty4 is a conjugate between the p458 peptide and theS. pneumoniae serotype 4 capsular polysaccharide; P458s-Ty4 is aconjugate between the p458s peptide and the S. pneumoniae serotype 4capsular polysaccharide.

REFERENCES

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What is claimed is:
 1. An isolated peptide antigen having an amino acidsequence selected from the group consisting of: LVTVNPFVSVATANS (SEQ IDNO: 11), LVTVNPFVSVATANA (SEQ ID NO: 12), YIVVGRGEQQINHHWHK (SEQ ID NO:21) and analogs, fragments, derivatives and salts thereof.
 2. Thepeptide antigen of claim 1, wherein the analogs have an amino acidsequence as set forth in any one of SEQ ID NOS: 25-44.
 3. A conjugatecomprising the peptide antigen of claim 1 covalently attached to acarrier capable of enhancing the immunogenicity of said peptide.
 4. Theconjugate of claim 3, wherein the carrier is selected from the group ofpeptides consisting of: (a) NEDQKIGIEIIKRTLKI (p458h; SEQ ID NO: 1), (b)NEDQKIGIEIIKRALKI (p458; SEQ ID NO: 2), (c) EGDEATGANIVKVALEA (p458mt;SEQ ID NO: 3), (d) NEDQNVGIKVALRAMEA (p458e; SEQ ID NO: 4), (e) ananalog or derivative of p458h (SEQ ID NO: 1) that has at least 70% ofthe electric and hydrophilicity/hydrophobicity characteristic of humanHSP60 from position 458 to position 474, said peptide, analog orderivative being capable of increasing substantially the immunogenicityof the viral antigen when the conjugate is administered in vivo,preferably wherein the carrier is an analog of p458h (SEQ ID NO: 1):⁴⁵⁸NEDQKIGIEIIKRTLKI⁴⁷⁴ in which the residue E⁴⁵⁹ is either E or D; theresidue D⁴⁶⁰ is either D or E; the residue K⁴⁶² is either K or R orornithine (Orn); the residue I⁴⁶³ is either I or L, V, M, F, norleucine(Nle) or norvaline (Nva); the residue I⁴⁶⁵ residue is either I or L, V,M, F, Nle or Nva; the residue E⁴⁶⁶ is either E or D; the residue I⁴⁶⁷ iseither I or L, V, M, F, Nle or Nva; the residue I⁴⁶⁸ is either I or L,V, M, F, Nle or Nva; the residue K⁴⁶⁹ is either K or R or Orn; theresidue R⁴⁷⁰ is either R, K or Orn; the residue L⁴⁷² in either L or I,V, M, F, Nle or Nva; the residue K⁴⁷³ is either K or R or Orn; and theresidue I⁴⁷⁴ is either I or L, V, M, F, Nle or Nva, (f)KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO: 76) and analogs and derivativesthereof, (g) KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO: 86) and analogs andderivatives thereof.
 5. The conjugate of claim 4 having an amino acidsequence as set forth in any one of SEQ ID NOS: 13-16 and 23-24 andanalogs, derivatives and salts thereof, or having an amino acid sequenceas set forth in any one of SEQ ID NOS: 56-64, or having an amino acidsequence as set forth in any one of SEQ ID NOS: 67-75 and 77-79.
 6. Anucleic acid sequence encoding the peptide of claim
 1. 7. The nucleicacid sequence of claim 6, wherein said sequence is as set forth in anyone of SEQ ID NOS: 19-20, 22 and 45-55.
 8. A vector comprising thenucleic acid sequence of claim 6 operably linked to one or moretranscription control sequences.
 9. A host cell comprising the vector ofclaim
 8. 10. A vaccine composition comprising a peptide antigenaccording to claim 1, further comprising at least one pharmaceuticallyacceptable carrier, adjuvant, excipient or diluent.
 11. The vaccine ofclaim 10 wherein the carrier is selected from the group consisting of:(a) NEDQKIGIEIIKRTLKI (p458h; SEQ ID NO: 1), (b) NEDQKIGIEIIKRALKI(p458; SEQ ID NO:2), (c) EGDEATGANIVKVALEA (p458mt; SEQ ID NO:3), (d)NEDQNVGIKVALRAMEA (p458e; SEQ ID NO:4), (e) an analog or derivative ofp458h (SEQ ID NO: 1) that has at least 70% of the electric andhydrophilicity/hydrophobicity characteristic of human HSP60 fromposition 458 to position 474, said peptide, analog or derivative beingcapable of increasing substantially the immunogenicity of the viralantigen when the conjugate is administered in vivo, (f)KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76) and analogs and derivativesthereof, (g) KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO:86) and analogs andderivatives thereof.
 12. A vaccine composition comprising a conjugateaccording to claim
 3. 13. A method for immunizing a subject in needthereof against a viral infection, comprising administering to thesubject a vaccine composition comprising a conjugate according to claim3.
 14. The method of claim 13 wherein the subject is selected from agroup consisting of: humans, non-human mammals and non-mammaliananimals.
 15. The method of claim 14, wherein said subject is human. 16.A method for immunizing a subject in need thereof against a viralinfection, comprising administering to the subject a vaccine compositioncomprising according to claim
 10. 17. A diagnostic kit comprising atleast one peptide antigen having an amino acid sequence as set forth inany one of SEQ ID NOS:11-12, 21 and 25-44 and analogs, homologs,derivatives and salts thereof, and means for determining whether thepeptide antigen binds specifically to a biological sample.
 18. A methodfor diagnosing exposure of a subject to a flavivirus or for diagnosing aflavivirus infection, comprising the steps of: (a) contacting a suitablebiological sample with a viral antigen having an amino acid sequence asset forth in any one of SEQ ID NOS:11-12, 21 and 25-44 and analogs,homologs, derivatives and salts thereof under conditions such that animmune reaction can occur; (b) determining whether the peptide antigenbinds specifically to the biological sample.
 19. The method according toclaim 18, wherein step (b) includes determining the extent ofantigen-antibody complex formation, wherein an antigen-antibody complexformation level significantly higher than the level obtained for asample obtained from a non-infected subject is indicative of exposure ofthe subject to the flavivirus.
 20. The method according to claim 18 forthe differential diagnosis of a flavivirus infection.